Nucleic acids encoding modified olfactory cyclic nucleotide gated ion channels

ABSTRACT

The present invention provides modified cyclic nucleotide gated (CNG) channels. In particularly preferred embodiments, the modified CNG channels exhibit increased sensitivity and specificity for cAMP, as compared to wild-type CNG channels. In additional embodiments, regulation by Ca 2+ -calmodulin has been removed in the modified CNG channels. Convenient optical methods for detecting changes in cAMP, taking advantage of the Ca 2+  permeability of the channel are also provided by the present invention. In addition, electrophysiological methods are further provided.

This application claims benefit of provisional patent application U.S.Ser. No. 60/332,494, filed on Nov. 16, 2001.

This invention was made with government support under grants GM32438,NS28389, HL58344, DC00385, EY09275, from the National Institutes Health.The United States Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention provides modified cyclic nucleotide gated (CNG)channels. In particularly preferred embodiments, the modified CNGchannels exhibit increased sensitivity and specificity for cAMP, ascompared to wild-type CNG channels. In additional embodiments,regulation by Ca²⁺-calmodulin has been removed in the modified CNGchannels. Convenient optical methods for detecting changes in cAMP,taking advantage of the Ca²⁺ permeability of the channel are alsoprovided by the present invention. In addition, electrophysiologicalmethods are further provided.

BACKGROUND OF THE INVENTION

Cyclic AMP (cAMP) is a ubiquitous intracellular second messenger thatcoordinates diverse cellular functions. It is produced in response to alarge variety of extracellular stimuli, including hormones andneurotransmitters. Typically, these agents bind to receptors on theextracellular surface, the receptors activate G-proteins on theintracellular surface and the G-proteins in turn activate adenylylcyclase, the enzyme that produces cAMP.

cAMP signals are considered to be complex, as evidenced by cAMP'sdifferential regulation of over 200 cellular targets. In addition, theenzymes involved in cAMP metabolism are known to be regulated bynumerous other signaling pathways. Unfortunately, as discussed furtherbelow, an understanding of cAMP signals has been elusive, due to thefact that current methods for measuring cAMP lack both temporal andspatial resolution. Thus, what is needed are high-resolution means thatprovided the requisite resolution in order to measure intracellularcAMP.

For example, the standard method for measuring cAMP accumulation withincells is to treat cells with [³H]-adenine to label the ATP pool, andthen measure the conversion of [³H]ATP to [³H]cAMP at different timepoints (See e.g., Evans et al., Mol. Pharmacol., 26:395–404 [1984]).This method is typically done on hundreds of thousands to millions ofcells. As a consequence, the method has no spatial resolution and itcannot be used to assess cell-to-cell variability within a population.In addition, it is labor-intensive because cAMP accumulation can bemeasured only at discrete time points (i.e., in contrast to fluorescenceor electrophysiological techniques, it does not provide a continuousreadout). This is an important consideration in screening applications.In addition, the method has low temporal resolution, as it isimpractical to measure cAMP accumulation in less than 5 secondincrements. Thus, it is likely that rapid changes in cAMP will be missedwhen this technique is used.

A second method currently used in the art involves measuring the changesin fluorescence energy transfer between labeled subunits ofcAMP-dependent protein kinase, which dissociate upon binding of cAMP.Fluorescent subunits are either prepared biochemically and microinjected(See, Adams et al., Nature 349:694–697 [1991]), or are geneticallyencoded (See, Zaccolo et al., Nat. Cell. Biol., 2:25–29 [2000]).Although this method does allow for detection of cAMP changes in singlecells, it has very low spatial resolution due to limitations in thewavelength utilized (i.e., it is limited by the wavelengths of visiblelight; 400–800 nm). It also has low temporal resolution, due to the slowreassociation of labeled subunits (i.e., a half-time of 100–200 secondsat typical subunit concentrations; See, Ogreid and Doskeland, Biochem.,22:1686–1696 [1983]), and the tendency of catalytic subunits thatcatalyze phosphorylation to accumulate in the nucleus (See, Harootunianet al., Mol. Cell. Biol., 4:993–1002 [1993]). In addition, in order tooverwhelm endogenous kinase, several micromolar labeled subunits areusually introduced into the test system. This strongly buffers naturalcAMP signals, and causes functional alterations of cellular targets dueto extensive phosphorylation. Thus, there remains a need in the art formethods and compositions that further elucidate the activities ofreceptors, G-proteins, phosphodiesterases (PDEs), adenylyl cyclases, andother proteins important in cAMP signalling.

SUMMARY OF THE INVENTION

The present invention provides modified CNG channels, in which thesensitivity and specificity for cAMP are increased. In addition,regulation by Ca²⁺-CaM is removed in the modified CNG channels.Convenient optical methods for detecting changes in cAMP, takingadvantage of the Ca²⁺ permeability of the channel are also provided bythe present invention.

In particular, the present invention provides isolated nucleic acidsencoding a modified olfactory cyclic nucleotide-gated ion channel,wherein the channel comprises mutations which together impart increasedcAMP sensitivity, decreased cGMP sensitivity, decreased nitric oxidesensitivity and decreased calcium-calmodulin sensitivity.

The present invention also provides isolated nucleic acids encoding amodified olfactory cyclic nucleotide-gated ion channel, in which thechannel comprises at least one mutation selected from the groupconsisting of a C460W mutation and a E583M mutation. In someembodiments, the channel comprises a E583M mutation; a C460W mutationand a E583M mutation; or a 61-90 deletion, a C460W mutation, and a E583Mmutation. In a subset of these embodiments, the channel comprises anamino acid sequence selected from the group consisting of SEQ ID NO:5,SEQ ID NO:6, and SEQ ID NO:7. Also provided in some embodiments arepolypeptides encoded by the isolated nucleic acids, and expressionvectors comprising the isolated nucleic acids. In preferred embodiments,the vector is a recombinant adenovirus vector. Furthermore, theinvention provides host cells comprising the expression vectors, inwhich the host cell is selected from the group consisting of aprokaryotic cell and a eukaryotic cell. In preferred embodiments, theeukaryotic cell is selected from the group consisting of a humanembryonic kidney-293 cell, a GH4C1 pituitary cell, and a rat C6-2Bglioma cell.

The present invention also provides methods for determining localintracellular cAMP concentration within an eukaryotic cell, comprisingthe steps of providing: at least one eukaryotic cell, and a nucleic acidencoding a modified olfactory cyclic nucleotide-gated ion channel, inwhich the channel comprises at least one mutation selected from thegroup consisting of a C460W mutation and a E583M mutation; andcontacting the eukaryotic cell with the nucleic acid under conditionssuitable for expressing the modified olfactory cyclic nucleotide-gatedion channel in the eukaryotic cell; measuring intracellular calciumconcentration in the eukaryotic cell; and determining localintracellular cAMP concentration based upon the intracellular calciumconcentration. In some embodiments, the contacting step comprisesinfecting the eukaryotic cell with a recombinant adenovirus comprisingthe nucleic acid. In some preferred embodiments, the measuring stepcomprises contacting the eukaryotic cell with a stimulus. In a subset ofthese embodiments, the stimulus is selected from the group consisting ofan adenylate cyclase activator, a G-protein activator, and aphosphodiesterase inhibitor. In particularly preferred embodiments, themeasuring step comprises monitoring calcium flux with a fluorescentcalcium indicator. Appropriate fluorescent calcium indicators includebut are not limited to fura-2, indo-1, quin-2, fluo-3 and rhod-2.

Moreover, the present invention provides methods for determining localintracellular cAMP concentration within an eukaryotic cell, comprisingthe steps of providing: at least one eukaryotic cell, and a nucleic acidencoding a modified olfactory cyclic nucleotide-gated ion channel,wherein the channel comprises at least one mutation selected from thegroup consisting of a C460W mutation and a E583M mutation; andcontacting the eukaryotic cell with the nucleic acid under conditionssuitable for expressing the modified olfactory cyclic nucleotide-gatedion channel in the eukaryotic cell; measuring the electric currentacross the plasma membrane of the eukaryotic cell; and determining localintracellular cAMP concentration based upon the electric current. Insome embodiments, contacting step comprises infecting the eukaryoticcell with a recombinant adenovirus comprising the nucleic acid. In somepreferred embodiments, the measuring step comprises contacting theeukaryotic cell with a stimulus. In a subset of these embodiments, thestimulus is selected from the group consisting of an adenylate cyclaseactivator, a G-protein activator, and a phosphodiesterase inhibitor. Insome embodiments, the measuring step comprises monitoring the electriccurrent with a patch-clamp technique selected from the group consistingof perforated patch-clamp technique and a whole-cell patch-clamptechnique. In related embodiments, the determining step comprisescalibrating the electric current with respect to cAMP concentration byobtaining a cAMP-dose response curve for the modified cyclicnucleotide-gated ion channel with an inside-outside patch clamptechnique.

Also provided by the present invention are methods for determiningwhether a candidate compound is capable of modulating localintracellular cAMP concentration within a eukaryotic cell, comprisingthe steps of providing: at least one eukaryotic cell expressing amodified olfactory cyclic nucleotide-gated ion channel, wherein thechannel comprises at least one mutation selected from the groupconsisting of a C460W mutation and a E583M mutation, and a drugcandidate; and determining local intracellular cAMP concentration withinthe eukaryotic cell in the presence and absence of the drug candidate.In some embodiments, the expressing of the modified olfactory cyclicnucleotide-gated ion channel is accomplished by infection of theeukaryotic cell with a recombinant adenovirus. In some preferredembodiments, the determining of the local intracellular cAMPconcentration is accomplished by measuring intracellular calciumconcentration in the eukaryotic cell in the presence and absence of thedrug candidate. In other preferred embodiments, the determining of thelocal intracellular cAMP concentration is accomplished by measuring theelectric current the plasma membrane of the eukaryotic cell in thepresence and absence of the drug candidate.

DESCRIPTION OF THE FIGURES

FIG. 1 provides the nucleotide sequence of a modified rat olfactorycyclic nucleotide-gated ion channel containing the E583M mutation (SEQID NO:1). The corresponding amino acid sequence is disclosed as SEQ IDNO:5.

FIG. 2 provides the nucleotide sequence of a modified rat olfactorycyclic nucleotide-gated ion channel containing the C460W/E583M mutation(SEQ ID NO:2). The corresponding amino acid sequence is disclosed as SEQID NO:6.

FIG. 3 provides the nucleotide sequence of a modified rat olfactorycyclic nucleotide-gated ion channel containing the Δ61-90/C460W/E583Mmutation (SEQ ID NO:3). The corresponding amino acid sequence isdisclosed as SEQ ID NO:7.

FIG. 4 provides the nucleotide sequence of the wild-type rat olfactorycyclic nucleotide gated channel (SEQ ID NO:4), GenBank Accession No. NM012928. The corresponding amino acid sequence is disclosed as SEQ IDNO:8.

FIG. 5 provides a quantitative description of the localized transientcAMP response and the total cellular cAMP accumulation. The inset ofthis Figure shows the two compartment model of the cell with adiffusional restriction between the membrane-localized microdomain(compartment 1) and the bulk cytosol (compartment 2). Panel A shows thatthe rapid activation of AC and slower activation of PDE shape thetransient signal in the microdomain. The slow flux of cAMP from themicrodomain allows low levels to accumulate in the cytosol. Note thateven in the small volume of the microdomain, the concentration of CNGchannels would be low (˜40 nM), and would not be expected to buffer themeasured cAMP signal. Panel B provides results showing that total cAMPlevels (microdomain and cytosol) reach a plateau. In this Figure, dashedlines indicate zero cAMP.

FIG. 6 provides a comparison of CNG channel constructs. Dose-responserelations of subunit homomultimers of WT (Panel A), E583M (Panel B),C460W/E583M (Panel C), and Δ61-90/C460W/E583M (Panel D) rat olfactoryCNG channels for cAMP (squares) and cGMP (triangles), evaluated at amembrane potential of +50 mV are shown. As indicated, the modified CNGchannels are more sensitive to cAMP than WT channels, and less sensitiveto cGMP. Solid lines are fits to the Hill equation. For the experimentsshown, K_(1/2) ^(cAMP)=30, 10.3, 1.4, and 15 μM; K_(1/2) ^(cGMP)=1.6,25, 11, and 36 μM for WT, E583M, C460W/E583M, and Δ61-90/C460W/E583Mchannels, respectively. Other fit parameters are included in Table 1,below.

FIG. 7 provides data showing forskolin-induced cAMP accumulation inHEK-293 cells monitored using Ca²⁺ influx through expressed CNGchannels. In these experiments, 100 μM IBMX was added at time zero anddifferent forskolin concentrations were added at 180 s (indicated by thearrows); concentrations (in micromolar) are indicated at the end of eachtrace. Panel A provides a graph showing the forskolin-induced Ca²⁺influx was observed in cells expressing the WT channel; the cAMPresponse (slope) did not saturate in the forskolin range tested (0–50μM). Panel B provides a graph showing that the forskolin-induced Ca²⁺influx was larger in cells expressing the E583M channel than WT channel(Panel A). More importantly, the responses saturated at 20 μM. Panel Cprovides a graph showing that the forskolin-induced responses in cellsexpressing the C460W/E583M channels saturated at a lower forskolinconcentration than the E583M channels (Panel B), at 5 μM. Panel Dprovides a graph showing forskolin-induced responses in cells expressingthe Δ61-90/C460W/E583M channel, saturating at 20 μM. For each construct,the variability in the response between batches of cells was <25% (n=4).

FIG. 8 provides a graph showing the typical forskolin-induced Ca²⁺influx in control HEK-293 cells. HEK-293 cells not expressing CNGchannel constructs were treated in the same manner as those describedabove for FIG. 7. Forskolin (0, 0.5, 5 or 50 μM) was added 180 s aftertreatment with 100 μM IBMX. As indicated, little or no Ca²⁺ influx wasobserved in control HEK-293 cells under any of the experimentalconditions described herein.

FIG. 9 provides data showing that PDE activity reduces forskolin-inducedCa²⁺ influx through CNG channels in HEK-293 cells. Ca²⁺ influx throughWT (Panel A), E583M (Panel B), C460W/E583M (Panel C), andΔ61-90/C460W/E583M (Panel D) CNG channels in response to 10 μM forskolin(180 s, indicated by arrows), after a 3 minute pre-treatment with eithervehicle (-IBMX) or 100 μM IBMX. Note that a robust forskolin-inducedresponse was easily monitored in the absence of IBMX using C460W/E583Mchannels as cAMP sensors (Panel C).

FIG. 10 provides data showing the effects of different PDE inhibitors onforskolin-induced Ca²⁺ influx in HEK-293 cells. C460W/E583M channelswere used to monitor cAMP accumulation in the presence and absence ofPDE inhibitors: A) 100 μM IBMX; B) 10 μM IBMX; C) 100 μM 8-MM-IBMX; D)10 μM EHNA; E) 1 μM trequinsin; F) 15 μM quazinone; G) 10 μM RO-20-1724;and H) 50 μM zaprinast. PDE inhibitors were added at time zero, and 1 μMforskolin was added at 180 s (indicated by arrows). The IC₅₀ for eachPDE inhibitor is provided in Table 3. As indicated in this Figure, onlythe non-specific PDE inhibitor IBMX (Panels A and B) and the PDE typeIV-specific inhibitor RO-20-1724 (Panel G) influenced the time course offorskolin-induced Ca²⁺ influx. In general, there was little variabilityin responses within a single batch of cells.

FIG. 11 provides data showing estimates of K_(I) for three different PDEinhibitors. As indicated, the results reveal that PDE type IV regulateslocal cAMP levels in HEK-293 cells. C460W/E583M channels were used tomonitor cAMP accumulation triggered by 0.5 μM forskolin (180 s) in thepresence and absence of PDE inhibitors (0 s). The d(ΔF/F₀)/dt valueswere determined as described below for FIG. 12. The linear fits used toestimate K_(I) values are shown in the insets. The K_(I)s of the PDEinhibitors were 10 μM (IBMX; Panel A), 0.14 μM (RO-20-1724; Panel B),and 0.09 μM (rolipram; Panel C) for the experiments shown.

FIG. 12 provides a graph showing the measurement of Ca²⁺ through CNGchannels in response to a rise in cAMP. Ca²⁺ influx caused a decrease influorescence (ΔF), which was expressed relative to the pre-stimulusfluorescence (F₀). In these experiments, HEK-293 cells expressingC460W/E583M CNG channels responded to increased cAMP levels caused byexposure to 50 nM rolipram added at 0 s (first arrow) and 0.5 μMforskolin added at 180 s (second arrow). After a brief lag, ΔF changedin a linear fashion (slope=6.2×10⁻⁴s⁻¹). Slopes were used to assessrelative Ca²⁺ influx rates, for estimating the K_(I) of PDE inhibitors(See, Eqs. 4 and 5).

FIG. 13 provides data showing detection of AC and PDE activity in GH4C1cells. Panel (A) provides data for GH4C1 cells expressing WT CNGchannels. Either 100 μM IBMX or vehicle (-IBMX; control) were added attime zero. Then, 50 μM forskolin was added at 180 s. Panel B providesdata for control cells (i.e., cells not expressing CNG channels). Noforskolin-induced Ca²⁺ influx was observed under these conditions, orany of the experimental conditions presented. Panels C and D provideresults for GH4C1 cells expressing C460W/E583M channels. IBMX or vehicle(-IBMX; control) was added at 60 s (first arrow). Either 10 μM forskolin(Panel C) or 100 nM VIP (Panel D) were added at 240 s (second arrow). Inthe absence of IBMX, large forskolin- or VIP-induced increases in Ca²⁺influx were observed. When the local PDE activity was inhibited by 100μM IBMX, substantial basal AC activity was revealed. This level of basalAC activity was quite different from that observed in HEK-293 cells(See, FIGS. 9 and 10). In these experiments, 1 μM nimodipine was addedat time 0 to block endogenous voltage-gated Ca²⁺ channels.

FIG. 14 provides data showing that PDE type IV is responsible forlocalized, high K_(m) PDE activity in GH4C1 cells. In these experiments,Δ61-90/C460W/E583M channels were used to detect changes in cAMPaccumulation in the presence and absence of PDE inhibitors. Inhibitorswere added at 60 s, 10 μM forskolin was added at 240 s, and 1 μMnimodipine was added at time zero (to block voltage-gated Ca²⁺channels). As indicated, the non-specific PDE inhibitor IBMX (Panel A),the PDE type 1-specific inhibitor 8-methoxymethyl-IBMX (8-MM-IBMX)(Panel B), the PDE type IV-specific inhibitors RO-20-1724 (Panel C), androlipram (Panel D) were shown to increase forskolin-induced Ca²⁺ influxthrough CNG channels.

FIG. 15 provides results from experiments to determine single-cellmeasurements of local cAMP signals. Upper portions of Panels A and Bshow that rapid application of PGE₁ triggered transient inward currentsthrough CNG channels (−20 mV). Two different cells were monitored inperforated patch configuration. The lower portions of Panels A and B thecorresponding cAMP signals calibrated as described herein. Panel Cprovides results showing that rapid application of PGE₁ triggered atransient inward current (whole cell configuration, −20 mV). Subsequentapplication of PGE₁ and IBMX triggered an inward current that rose to aplateau. This current was blocked by 10 mM MgCl₂ (characteristic of CNGchannels). Dashed lines indicate either zero cyclic nucleotide-inducedcurrent (the current in 10 mM MgCl₂) or zero cAMP. No PGE₁-inducedcurrents were observed in cells not expressing CNG channels. Severaladditional controls were done to ensure that the PGE₁-induced signal wasdue to a rise and fall in cAMP. PGE₁ had no direct effect on channels inexcised membrane patches, and when it was applied to cells it did notaffect the cAMP sensitivity, the conductance, nor the number of activechannels. In addition, treatment of CNG-channel-expressing cells withPGE₁ triggered little or no release of Ca²⁺ from internal stores and nomeasurable increases in cGMP, as shown in FIGS. 18 and 19.

FIG. 16 provides a comparison of membrane-localized (A) and total cAMP(B) levels in cell populations. In Panel A, shows that sustainedapplication of 10 μM PGE₁ in the absence of PDE inhibitor caused anincrease in Ca²⁺ influx, followed by a decline in Ca²⁺. Little or noincrease in Ca²⁺ was observed in cells not expressing the channel. Theinitial interpretation was that PGE₁ triggered a rise and fall in localcAMP concentration: the rise caused an increase in Ca²⁺ influx throughCNG channels; the subsequent fall in cAMP led to reduced Ca²⁺ influx;and, Ca²⁺ pumping mechanisms caused the decline in Ca²⁺ levels. Insupport of this interpretation, PGE₁ in the presence of the PDEinhibitor IBMX caused Ca²⁺ to rise along a similar time-course, but thedecay phase was abolished. These results indicate that the underlyingcause of the decay phase was hydrolysis of cAMP. In populations of cellsexpressing C460W/E583M channels, PGE₁ caused total cellular cAMPaccumulation, assessed as the conversion of [³H]ATP to [³H]cAMP, to riseto a plateau in the absence of IBMX (See, FIG. 16, Panel B). This is inmarked contrast to the transient increase in cAMP inferred from FIG. 16,Panel A.

FIG. 17 provides results showing that PGE₁ triggers sustained AC and PDEactivity. Panels A and B show local cAMP changes in response to ACstimulation, monitored by Ca²⁺ influx through C460W/E583M channels. PGE₁(Panel A) or forskolin (Panel B) were applied at the indicatedconcentrations. Panel C shows rises in cAMP (monitored by Ca²⁺ influx)caused by 100 nM PGE₁ (t=0) and subsequent addition of 100 μM IBMX (1,2, 5, 10, 20, or 50 min; superimposed traces). The slopes of theIBMX-induced responses (0.0044±0.0008 s⁻¹) were similar to each otherand to the slopes of the initial PGE₁-induced responses (0.0038±0.0003s⁻¹). IBMX and RO-20-1724 had no effect on channel activity in excisedmembrane patches (Rich et al. [2001], supra). Panel D provides resultsshowing that the second additions of 100 nM PGE₁ (2, 5, 10, 20, or 50min; superimposed traces) following the initial addition of 100 nM PGE₁(t=0) gave little or no response.

FIG. 18 provides data showing that PGE₁ treatment of HEK-293 cells doesnot alter CNG channel properties. To ensure that PGE₁ was not alteringthe number of channels, conductance, or cAMP sensitivity, severalcontrol experiments were performed on cells expressing C460W/E583Mchannels. Panel A provides results indicating a typical cAMPdose-response relation in an excised membrane patch, from a cell thatwas treated with 1 μM PGE₁ for >5 min prior to patch excision (circles).Patches were excised directly into a solution containing 1 μM cAMP (asubsaturating concentration). Currents were monitored for >30 min afterpatch excision, and no change in channel activity was observed. Therewas no significant difference in cAMP sensitivity between patchesexcised from cells pretreated with PGE₁ (K_(1/2)=1.2 μM, solid line) andthose excised from control cells (i.e., cells not pretreated with PGE₁;K_(1/2)=1.1 μM, dotted line). To test whether PGE₁ directly modulatedchannel properties, excised, inside out patches were exposed to PGE₁. Atypical example is shown in Panel B. A patch exposed to 1 μM cAMP wastreated for the indicated period with 1 μM PGE₁; no change in currentwas observed (+50 mV). Panel C provides results from experiments todetermine whether PGE₁ could alter the number of active channels. Inthese experiments, currents were monitored in the whole cellconfiguration (+50 mV) with 100 μM 8-(p-chlorophenylthio)-cGMP (asaturating concentration of a hydrolysis-resistant cGMP analog) in thepatch pipette. In order to ensure that the nucleotide had equilibratedthroughout the cell, there was a waiting period of more than 10 minafter break-in to ensure that the nucleotide had equilibrated throughoutthe cell. Currents were unaffected by extracellular application of 1 μMPGE₁. The leak current was determined by blocking the channels with 10mM MgCl₂.

FIG. 19 provides results showing that PGE₁ does not trigger significantCa²⁺ release from internal stores nor production of membrane localizedcGMP, in HEK-293 cells expressing CNG channels. Changes in internal Ca²⁺were monitored using fura-2 as described herein. Panel A providesresults for cells expressing C460W/E583M CNG channels that wereresuspended in nominally Ca²⁺ free solution (−Ca²⁺ trace) one minuteprior to the addition of 100 mM PGE₁ (arrow). PGE₁ caused little or norise in Ca²⁺, indicating negligible release from internal stores. As apositive control for CNG channel expression, the experiment was repeatedin the presence of 1 mM Ca²⁺ using an aliquot from the same batch ofcells (+Ca²⁺ trace) a large transient Ca²⁺ influx through CNG channelswas observed. Panel B provides results showing that cGMP did notcontribute to the observed transient responses in experiments whichmonitored Ca²⁺ influx (1 mM external Ca²⁺) through wild-type (WT)olfactory CNG channels (which have a significantly higher sensitivity tocGMP and a significantly lower sensitivity to cAMP than C460W/E583Mchannels (Rich et al. [2001] supra). After the addition of 100 nM PGE₁there was no observable Ca²⁺ influx. This demonstrates that cGMP made nocontribution to the PGE₁-induced responses measured with thehigh-cAMP-affinity C460W/E583M channels. As a positive control for WTCNG channel expression, (100 μM) forskolin-induced Ca²⁺ influx wasmonitored following a 3-min pretreatment with 100 μM IBMX.

DESCRIPTION OF THE INVENTION

The present invention provides modified cyclic nucleotide gated (CNG)channels. In particularly preferred embodiments, the modified CNGchannels exhibit increased sensitivity and specificity for cAMP, ascompared to wild-type CNG channels. In additional embodiments,regulation by Ca²⁺-calmodulin has been removed in the modified CNGchannels. Convenient optical methods for detecting changes in cAMP,taking advantage of the Ca²⁺ permeability of the channel are alsoprovided by the present invention. In addition, electrophysiologicalmethods are further provided.

In some preferred embodiments, the present invention providesgenetically-modified cyclic nucleotide-gated ion channels. This class ofchannels is directly opened by the binding of cyclic nucleotides. Thesechannels were discovered in retinal photoreceptor cells and olfactoryreceptor neurons, where they generate the electrical response to lightand odorants. The native retinal channel is cyclic GMP (cGMP) specific,while the native olfactory channel is equally sensitive to cAMP andcGMP. Native channels consist of both and subunits, both of which bindcyclic nucleotides. Open channels allow cations (Na⁺, K⁺, Ca²⁺, Mg²⁺) topass through the cell membrane. Thus, the activation of channels isreadily detected with both electrophysiological and Ca²⁺-imagingtechniques, as known in the art.

As discussed in greater detail herein, cyclic nucleotide-gated (CNG)channels have several properties that make them attractive as cAMPsensors. First, they incorporate into discrete regions of the surfacemembrane where adenylyl cyclase is known to reside. Second, severalhundred channels in a cell give a robust signal without significantlybuffering the cAMP being measured. Third, cyclic nucleotide-gatedchannels respond rapidly to changes in cyclic nucleotide concentration.Fourth, the sensor can be calibrated in different cell types bymeasuring the apparent cAMP affinity in excised membrane patches. Thiscontrols for the possible modulation of CNG channels by endogenousproteins.

Retinal and olfactory α subunits form functional channels on their own,and the rat olfactory α subunit was previously used to measure cAMPaccumulation under strong stimulus conditions. However, the wild-type αsubunit has several drawbacks for measuring physiologic cAMP signals.First, wild-type channels (See, FIG. 4, and SEQ ID NO:4) have a lowapparent affinity for cAMP, which makes it difficult to detect the lowcAMP concentrations that activate protein kinase A (PKA), a crucialeffector of cAMP signals. Second, these channels are much more sensitiveto cGMP than cAMP. The K_(1/2) values (i.e., the concentrations thathalf-maximally activate the channel) are 1.6 and 36 μM, respectively(See, Table 1, below). Third, they can also be activated directly bynitric oxide. Fourth, the binding of the Ca²⁺-calmodulin (Ca²⁺-CaM)complex to these channels strongly inhibits channel opening. To overcomethese limitations, the present invention provides novel CNG channelswith improved cAMP-sensing capabilities.

In some embodiments of the present invention, a mutant rat olfactorychannel α subunit is used. In order to create the mutant, modificationswere made to the rat olfactory channel α subunit to increase itssensitivity and specificity for cAMP and to remove regulation byCa²⁺-CaM. In particular, new mutations, designated as “C460W” and“E583M” were used to produce a channel with significantly increasedsensitivity and specificity for cAMP. The resultant C460W/E583M channelhas K_(1/2) values of 1.2 and 12 μM for cAMP and cGMP (See, Table 1,below). To remove regulation of the channel by Ca²⁺-CaM binding residues61 through 90 were deleted.

The Δ61-90/C460W/E583M channel has two important advantages. First, itis virtually insensitive to cGMP. The current elicited by a saturatingconcentration of cGMP is 16% of the current elicited by saturating cAMP(See, Table 1, below). In addition, 36 μM cGMP is required to elicithalf of that tiny response. Second, the channel is not inhibited byCa²⁺-CaM. The sensitivity to cAMP is reduced (K_(1/2)=14.5 μM), whichallows cAMP to be measured in the upper end of the physiologic range.The use of both mutant channels allows cAMP to be measured over theentire physiologic range (0.1 to 50 μM).

As described in greater detail herein, the effects of modifications toCNG channels were assessed using the Hill equation,I/I_(max)=[cNMP]^(N)/([cNMP]^(N)+K_(1/2) ^(N)), where I/I_(max) is thefraction of maximal current, cNMP represents cyclic nucleotide, K_(1/2)is the concentration that gives half-maximal current, and N is the Hillcoefficient, an index of cooperativity. I′_(max) ^(cGMP)/I_(max) ^(cAMP)is the current induced by saturating cGMP divided by the current inducedby saturating cAMP. Data (mean±standard deviation of three experiments)were obtained from excised membrane patches using standard patch clamptechniques.

In particularly preferred embodiments, the coding sequence of thechannels have been incorporated into adenovirus constructs, which allowfor efficient expression in a variety of cell types. The adenovirusconstructs of preferred embodiments of the present invention are deletedfor the E3 region and have the E1 region replaced by the CNG channelalleles under the control of the cytomegalovirus major intermediatepromoter. The channels have been used to measure cAMP in two differentassay designs.

One assay system involves a convenient, optical method for detectingchanges in cAMP, which takes advantage of the Ca²⁺ permeability of thechannel. There are several advantages of measuring Ca²⁺ influx throughCNG channels as a way to monitor changes in cAMP. Importantly, thisassay is very simple to implement in either cell populations or singlecells. In addition, the measurement has a high signal-to-noise ratio.Furthermore, it is sensitive to small changes in adenylyl cyclaseactivity. In contrast to the present invention, in traditional assays,strong stimuli and phosphodiesterase (PDE) inhibitors are often requiredto detect cAMP accumulation. Natural cAMP signals are severely distortedby the necessity to inhibit PDE, the enzyme responsible for degradingcAMP. In contrast to the currently used means, the novel channelsdescribed herein readily detect changes in cAMP in response tophysiologic stimuli, and in the absence of PDE inhibitors. This assay isparticularly well-suited as an initial screen for changes in cAMP.

Another assay system that finds use with the present invention involvesthe use of electrophysiological assays, in which either perforated patchor whole-cell configurations of the patch clamp technique are used tomeasure currents through the ion channels. This assay is particularlyappropriate when more precise measurements of local cAMP are required.These measurements take advantage of the fast response of CNG channelsto changes in cAMP concentration, without the distortions that arisefrom the Ca²⁺ handling properties of the cell. The higher dynamic range,increased temporal resolution, and more accurate measurement of cAMPconcentration provided by these methods find use in providinginformation regarding cAMP signals and signalling systems. The increasedsensitivity of the novel CNG channels of the present invention wereconfirmed using in vivo assays by monitoring cAMP-induced Ca²⁺ influxthrough the channels in cell populations and by measuring cAMP-inducedcurrents in the whole-cell patch clamp configuration.

Further, as discussed herein, the present invention provides methods andcompositions for measuring changes in intracellular cAMP. A very largenumber of natural agents (e.g., hormones, neurotransmitters, odorants)and drugs bind to G-protein coupled receptors and cause changes in cAMPlevels. As the present invention provides a system with demonstratedrobust expression of the adenovirus-encoded sensor in several celltypes, the present invention provides means for both high-throughputpharmacological screens as well as for many research purposes.

Indeed, the compositions of the present invention have been used: 1) todemonstrate the existence of distinct cAMP signals in different regionsof a simple cell; 2) to probe the interplay between stimulatory andinhibitory G-protein regulation of adenylyl cyclase; 3) to identify thephosphodiesterase (PDE) subtypes that shape cAMP levels in non-excitableand excitable cells (these experiments also provided the inhibitionconstants of several PDE inhibitors in the intact cell). The presentinvention provides sensors that have a high signal-to-noise ratio overmost of the physiologic range of cAMP concentrations (100 nM to 100 μM).However, it is contemplated that additional sensors of the presentinvention will find use over the entire range of physiological cAMPconcentrations (i.e., down to 20 nM).

As discussed in greater detail elsewhere herein, in some particularlypreferred embodiments, the present invention provides compositionscomprising several modifications to the WT olfactory CNG channel thatsignificantly enhance its utility as a cAMP sensor. Testing of threenovel constructs is described herein. As discussed in greater detailherein, in comparison with WT CNG channels, the E583M channel (SEQ IDNO:5) is more sensitive to cAMP and less sensitive to cGMP. However, twoadditional mutations produced channels that make even better cAMPsensors. The C460W/E583M channel (SEQ ID NO:6) has a high affinity forcAMP, allowing it to detect cAMP at levels which activate PKA. TheΔ61-90/C460W/E583M channel (SEQ ID NO:7) is almost as sensitive to cAMPas the E583M channel. However, the Δ61-90/C460W/E583M channel has twoimportant advantages: it is not inhibited by Ca²⁺-CaM, and it is barelyactivated by cGMP. Thus, the mutant CNG channels provided by the presentinvention provide the first ion channels that have been tailored formeasurement of cAMP.

The present invention also provides convenient assay systems fordetecting changes in local cAMP concentrations and examining PDEactivities. PDEs, the crucial terminators of cAMP and cGMP signals, werediscovered almost 40 years ago (Drummond and Perrot-Yee, J. Biol. Chem.,236:1126–1129 [1961]). Since then, PDEs have been classified into elevenfamilies according to substrate specificity, regulation, pharmacology,and more recently, amino acid homology (Beavo, Adv. Second MessengerPhosphoprot. Res., 22:1–38 [1988]; Beavo, Physiol. Rev., 75:725–748[1995]; Conti et al., Endocrine Res., 16:370–389 [1995]). Studies haveconfirmed differential regulation of PDE families by Ca²⁺-calmodulin(Ca²⁺-CaM), G-proteins, phosphorylation, and cyclic nucleotides. Thediversity of PDE families has led to the realization that PDE activityis a central element in the control of second messenger signaling, asimportant as adenylyl and guanylyl cyclase activity in shaping cyclicnucleotide signals. Yet, little is known about how PDE regulates cyclicnucleotide signals in vivo or how cyclic nucleotide signalsdifferentially regulate hundreds of cellular targets because, outside ofspecialized cells, there are few convenient real-time measures of cyclicnucleotide concentration. However, the present invention provides themeans to address these questions and obtain real-time measurements ofcyclic nucleotide concentrations.

In some particularly preferred embodiments, both WT and modified CNGchannels are used. There are several advantages provided by the methodsand compositions of the present invention involving measuring Ca²⁺influx through CNG channels as a way to monitor changes in cAMP. First,this assay is very simple to implement in either cell populations orsingle cells. Second, the measurement has a high signal-to-noise ratio.Third, it is sensitive to small changes in AC activity. This is due, inpart, to the fact that CNG channels detect local rather than totalcellular cAMP. With traditional assays, high agonist and PDE inhibitorconcentrations are often required to detect cAMP accumulation. Incontrast, the channels provided by the present invention readily detectchanges in cAMP caused by subsaturating agonist concentration in theabsence of PDE inhibitors. In addition to being easy to use, the presentinvention provides improved spatial and temporal resolution, as well asmeans for calibration to provide precise measures of cAMPconcentrations. Thus, among other utilities, the assay systems presentedherein find use as initial screens for changes in cAMP in response tophysiological stimuli and/or pharmacological agents.

The present invention provides significant improvements in assaysystems. However, in some cases, it is difficult to extract the precisecAMP concentration from these assay systems because the responses cannotbe calibrated (i.e., with known intracellular cAMP), and because Ca²⁺handling and depletion of free fura-2 become issues at high Ca²⁺ influxrates. Nonetheless, any of these limitations can be overcome when moreprecise measurements of local cAMP are required, by determining the cAMPconcentration using electrophysiological methods as known in the art(Rich et al. [2000], supra).

During the development of the present invention, observations were madeusing HEK-293 and C6-2B glioma cells, as well as GH4C1, that CNGchannels measure cAMP produced in subcellular compartments ormicrodomains near the plasma membrane, and that diffusion of cAMPbetween the microdomains and the bulk cytosol is severely hindered (See,Rich et al. [2000], supra). This conclusion was based on several linesof evidence. First, forskolin-induced increases in cAMP concentrationmeasured using CNG channels (>25 μM) were much greater than theincreases in cAMP concentration averaged throughout the accessible cellvolume (1–2 μM). Second, forskolin-induced cAMP accumulation was easilydetected by CNG channels in the rapidly-dialyzed, whole-cell patch clampconfiguration. Third, the wash-in of cAMP from the patch pipette to theCNG channels was much slower than would be expected based upon the rapidexchange of the bulk cytosol. All of these results were described by athree-compartment model (microdomain, cytosol, whole-cell pipette) inwhich the transfer rates between compartments were determined using datafrom the wash-in experiments.

During the development of the present invention, much was learned aboutthe two dimensional localization of proteins involved in cellularsignaling. For example, certain isoforms of PDE are differentiallydistributed between particulate (membrane-localized) and supernatant(cytosolic) cellular fractions. In addition, PDE kinetics may vary withcellular localization (See, Bolger et al., Biochem. J., 328:539–548[1997]). Elements of G-protein signaling pathways have been shown topreferentially localize within caveolae (i.e., distinct regions of theplasma membrane; See e.g., Rybin et al., J. Biol. Chem., 275:41447–41457[2000]). Furthermore, a recent report has described the localization ofdifferent voltage-gated K⁺ channels to distinct populations of lipidrafts (Martens et al., J. Biol. Chem., 276:8409–8414 [2001]).

However, two dimensional localization within the plasma membrane doesnot account for the three dimensional compartmentalization of cAMPsignals. Data obtained during the development of the present inventionshow that proximity to AC does not provide sufficient cAMPconcentrations for activation of effector proteins because, in essence,each cAMP molecule diffuses away more rapidly than the next molecule issynthesized. Thus, PDE activity alone is unlikely to be responsible forcompartmentalized cAMP signals, because PDE activity only lowers cAMPlevels. Thus, it is contemplated that PDEs regulating cAMP levelsdetected by CNG channels are localized within the same three dimensionalcompartment as AC and CNG channels. Importantly, by regulating cAMPconcentration within this compartment, PDE activity affects the rate ofcAMP flux between compartments. This PDE is contemplated to be acomponent of the particulate PDE fractions. In addition, cytosolic PDEis contemplated to regulate cAMP in other cellular compartments.

During the development of the present invention, Ca²⁺ influx through CNGchannels was used to detect changes in cAMP concentration in bothnonexcitable HEK-293 cells and excitable pituitary GH4C1 cells. One goalwas to assess the PDE subtypes responsible for shaping cAMP signals nearthe plasma membrane. In order to study the effects of PDEs, it isimportant to be able to first detect cAMP changes in the absence of PDEinhibitors. There was no difficulty with this in either cell type,despite the modest levels of cAMP accumulation in response to forskolinor VIP stimulation reported previously (Mollard et al., Biochem. J.,284:637–640 [1992]; Fagan et al., J. Biol. Chem., 271:12438–12444[1996]; and Fagan et al., J. Biol. Chem., 275:40187–40194 [2000]). InHEK-293 cells, only nonselective and PDE-type-IV selective inhibitorsdecreased PDE activity. The in vivo estimates of K_(I) for three PDEinhibitors (IBMX, RO-20-1724, and rolipram) described herein, areconsistent with IC₅₀ values estimated in vitro for PDE type IV. Thesedata are also consistent with a previous report that identified twoisoforms of PDE type IV (PDE4D3 and PDE4D5) in HEK-293 cells based onWestern blot analysis (Hoffmann et al., EMBO J., 18:893–903 [1999]).Inhibition of PDE activity did not reveal basal AC activity in HEK-293cells. In GH4C1 cells, inhibitors selective to PDE types I and IVdecreased PDE activity. Using the high cAMP affinity CNG channels,inhibition of PDE type I activity led to a rise in local cAMPconcentration in the absence of AC stimulation. This increase revealedsubstantial basal AC activity in GH4C1 cells. Under the same conditions,no change in cAMP level was observed when PDE type IV inhibitors wereadded. However, using the lower cAMP affinity, Δ61-90/C460W/E583Mchannels, inhibition of either PDE type I or type IV increasedforskolin-induced cAMP accumulation. These observations point to thepresence of two different PDE types: a low K_(m), PDE type I, and ahigher K_(m), PDE type IV. Based on the apparent cAMP affinities of thetwo channel constructs, the two K_(m) values were estimated to be <1 μMand >5 μM.

Interestingly, the data in GH4C1 cells indicate the presence of a futilecycle of cAMP synthesis and hydrolysis. Restricting this phenomenon tosubcellular compartments would be advantageous energetically, but thequestion remains as to why the additional energy is expended by thecells. One possibility is that constant AC and PDE activity allow thesystem to respond rapidly to a stimulus. In essence, the enzymes arepoised to respond to changes in active G-protein or changes in internalCa²⁺, allowing for rapid increases or decreases in cAMP levels. Asimilar situation exists in light-adapted photoreceptor cells (See e.g.,Nikonov et al., J. Gen. Physiol., 116:795–824 [2000]). Nonetheless, anunderstanding of the mechanism(s) is not necessary in order to use thepresent invention.

The existence of PDE type I in GH4C1 cells is consistent withobservations showing that CaM antagonists inhibit the hydrolysis of cAMP(Sletholt et al., Acta. Physiol. Scand., 130:333–343 [1987]).Furthermore, a high K_(m) (28.6 μM) and a low K_(m) (0.66 μM) PDE havebeen identified in both GH3 and GH4C1 cells, each PDE with a differentIC₅₀ for theophylline (Gautvik et al., Mol. Cell. Endocrinol.,26:295–308 [1982]). These K_(m) values are consistent with the in vivoestimates of the K_(m) values for PDEs that regulate local cAMP levelsdiscussed herein. The presence of both high and low K_(m) PDEs offerstwo possibilities for the shaping of cAMP signals: (1) that both PDEtypes regulate cAMP levels within a single compartment, or (2) that theydifferentially regulate cAMP levels in different cellular compartments.

If the two PDE types coexist in the same subcellular compartment thenthe low K_(m) PDE would be likely to regulate cAMP levels under basalconditions. Upon further activation of AC, this PDE would beoverwhelmed, and large concentrations of cAMP would accumulate locally.The high K_(m) PDE would be able to efficiently hydrolyze cAMP at theseelevated concentrations. Furthermore, it is contemplated that these PDEtypes are differentially regulated, allowing different feedbackmechanisms to control cAMP levels within this compartment.

If the activity of the two PDE types is compartmentalized, it iscontemplated that distinct cyclic nucleotide signals are capable ofoccurring simultaneously in different regions of the cell. Indeed, thispossibility seems likely because cAMP can be excluded from areas of thecell by PDE activity (See, Jurevicius and Fischmeister, Proc. Natl.Acad. Sci. USA 93:295–299 [1996]), cAMP is produced in microdomains withrestricted diffusional access to the bulk cytosol and different pools ofcGMP have different functional effects in ECV304 epithelial cells (Zolleet al., J. Biol. Chem., 275:25892–25899 [2000]).

It is contemplated that the present invention will find use in measuringlocal cAMP signals in response to a variety of extracellular stimuli.Indeed, the present invention provides means to investigate how cAMP iscapable of differentially regulating more than 200 cellular targets.This requires a quantitative knowledge of how cAMP signals are initiatedand terminated. In addition, different PDE subtypes are likely to playcrucial roles in shaping the signals in different cellular compartments.Within these compartments, cAMP levels are likely to vary dynamically(Brooker, Science 182:933–934 [1973]; Cooper et al., Nature 374:421–424[1995]; and Cooper et al., Adv. Second Messenger Phosphoprotein Res.,32:23–51 [1998]), particularly in excitable cells, where Ca²⁺ levelsoscillate during a train of action potentials. For example, it iscontemplated that modulation of PDE type I by Ca²⁺-CaM in excitableGH4C1 cells contributes to transient or oscillating cAMP signals. Ingeneral, dynamic cAMP signals would escape detection with conventionaltechniques.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides modified cyclic nucleotide gated (CNG)channels. In particularly preferred embodiments, the modified CNGchannels exhibit increased sensitivity and specificity for cAMP, ascompared to wild-type CNG channels. In additional embodiments,regulation by Ca²⁺-calmodulin has been removed in the modified CNGchannels. Convenient optical methods for detecting changes in cAMP,taking advantage of the Ca²⁺ permeability of the channel are alsoprovided by the present invention. In addition, electrophysiologicalmethods are further provided.

Retinal rod outer segments are the most closely examined secondmessenger signaling system, in part because the endogenous cyclicnucleotide-gated (CNG) channels have been used as real-time detectors ofcGMP concentration (Baylor et al., J. Physiol., 288:589–611 [1979]).Information from biochemical measurements of guanylyl cyclase andcGMP-specific PDE have been combined with real-time measurements of cGMPconcentration to characterize the relationship between these enzymes inregulating cGMP levels (See e.g., Fung et al., Proc. Natl. Acad. Sci.USA 78:152–156 [1981]; Koch and Stryer, Nature 334:64–66 [1988];Gorczyca et al., Proc. Natl. Acad. Sci. USA 91:4014–4018 [1994];Koutalos et al., J. Gen. Physiol., 106:891–921 [1995a]; Koutalos et al.,J. Gen. Physiol., 106:863–890 [1995b]; He et al., Neuron 20:95–102[1998b]; Tsang et al., Science 282:117–121 [1998]; Chen et al., Nature403:557–560 [2000]Leskov et al., Neuron 27:525–537 [2000]; and Nikonovet al., J. Gen. Physiol., 116:795–824 [2000]). The convergence ofdifferent approaches has led to an unprecedented understanding offeedback signaling within this model system (See e.g., Stryer, J. Biol.Chem., 266:10711–10714 [1991]; Lagnado and Baylor, Neuron 8:995–1002[1992]; Pugh and Lamb, Biochim. Biophys. Acta 1141:111–149 [1993];Yarfitz and Hurley, J. Biol. Chem., 269:14329–14332 [1994]; Yau, Invest.Ophthalmol. Vis. Sci., 35:9–32 [1994]; Polans et al., Trends Neurosci.,19:547–554 [1996]; Pugh et al., Biosci. Rep., 17:429–473 [1997]; andMolday, Invest. Ophthalmol. Vis. Sci., 39:2493–2513 [1998], for review).Nonetheless, an understanding of the mechanism(s) is not necessary inorder to use the present invention.

During the development of the present invention, it was determined thatcAMP concentration can be measured accurately with the rat wild-type(WT) olfactory cyclic nucleotide-gated (CNG) channel (See, FIG. 4 andSEQ ID NO:4), encoded by an adenovirus vector (See, Rich et al., J. Gen.Physiol., 116:147–161 [2000]). An important finding in multiple celltypes was that cAMP is produced in subcellular compartments near theplasma membrane. Diffusion between these compartments and the bulkcytosol is severely restricted. These regions likely allow for rapid andenergetically-efficient activation of cAMP-dependent processes, and itis contemplated that these relationships help explain how cAMP candifferentially regulate large numbers of cellular targets. However, asindicated above, an understanding of the mechanism(s) is not necessaryin order to use the present invention.

As described in greater detail herein, the present invention providesmodified CNG channels to increase its sensitivity and specificity forcAMP and to remove regulation by Ca²⁺-CaM. The present invention furtherprovides convenient optical methods for detecting changes in cAMP,taking advantage of the Ca²⁺ permeability of the channel. Using themodified CNG channels, this assay is sensitive to cAMP in thephysiologic range (0.1–50 μM). As described herein, this approach hasbeen used to probe the interactions between adenylyl cyclase (AC) andPDE in regulating local cAMP concentration in two cell types,nonexcitable HEK-293 and excitable GH4C1 pituitary cells.

cAMP and Signalling

Much has been learned about the enzymes involved in the generation andbreakdown of cAMP (Sunahara et al., Ann. Rev. Pharmacol. Toxicol.,36:461–480 [1996]; and Beavo, Physiol. Rev., 75:725–748 [1995]), and theproteins that mediate the downstream effects of cAMP (Walsh et al., J.Biol. Chem., 243:3763–3765 [1968]; Gray et al., Curr. Opin. Neurobiol.,8:330–334 [1998]; Francis and Corbin, Crit. Rev. Clin. Lab. Sci.,36:275–328 [1999]; and Finn et al., Ann. Rev. Physiol., 58:395–426[1996]). However, conventional methods have, by and large, been unableto resolve the spatial and temporal features of cAMP signals. Creativeattempts to use protein kinase A (PKA) or L-type Ca²⁺ channels to assesschanges in cAMP levels have yielded some information. In largeinvertebrate neurons gradients in cAMP between the processes and thesoma have been inferred (Bacskai et al., Science 260:222–226 [1993]; andHempel et al., Nature 384:166–169 [1996]). Gradients in cAMP have alsobeen deduced in studies of cardiac myocytes, in which only part of thecell was stimulated with isoproterenol (Jurevicius and Fischmeister,Proc. Natl. Acad. Sci. USA 93:295–299 [1996]). Unfortunately, thesemethods have limited resolution. It has been shown previously thatheterologously-expressed, cyclic nucleotide-gated (CNG) channels areable to detect changes in membrane-localized cAMP concentration (Rich etal., J. Gen. Physiol., 116:147–161 [2000]; Rich et al., J. Gen.Physiol., 118:63–77; and Fagan et al., FEBS Lett., 500:85–90 [2001])and, using the patch-cram technique, cytosolic cGMP (Trivedi and Kramer,Neuron 21:895–906 [1998]) in real time.

In contrast, in some embodiments of the present invention, theproperties of CNG channels are tailored for the measurement ofsubmicromolar cAMP concentrations (Rich et al. [2001], supra). Thesemethodological advances allow the measurement of cAMP produced by lowlevels of adenylyl cyclase (AC) activity without the need to inhibitphosphodiesterase (PDE) (Rich et al. [2001], supra; and Fagan et al.,supra).

During the development of the present invention, comparisons of themeasurements of membrane-localized and total cellular cAMP levels inresponse to prostaglandin E₁ (PGE₁) stimulation were made. The olfactoryCNG channel α subunit containing two mutations, C460W and E583M, wereused to monitor cAMP levels near the surface membrane in both cellpopulations and single cells. These mutations make it possible tomeasure cAMP concentrations in the 100 nM range, while rendering thechannel relatively insensitive to cGMP. Total cAMP accumulation in cellpopulations was investigated by measuring the conversion of [³H]ATP into[³H]cAMP. The difference between the two measured cAMP signals wasstriking: a rise and fall in cAMP near the membrane and an increase to asteady level throughout the cell. The segregation of signals helps toexplain how cAMP can differentially regulate cellular targets.

Indeed, results provided herein indicate that cAMP signals near thechannels are distinct from those in other parts of the cell. Severallines of evidence presented previously suggest that cAMP is produced insubcellular compartments near the surface membrane, and that diffusionbetween these domains and a cytosolic compartment is significantlyimpeded (Rich et al., [2000], supra). At a minimum, it is necessary toinvoke the same two compartments to explain the current data. With norestriction on diffusion: 1) cAMP concentrations right next to AC arenot high enough to activate PKA (Rich et al. [2000], supra), let alonethe CNG channels used here (unless the entire cell filled with cAMP);and 2) cAMP would diffuse across a 15–20 μm cell in <0.2 s, which would‘wash out’ any spatial differences on a time-scale of tens to hundredsof seconds. The inset to FIG. 5, Panel A shows the two compartment modelpresented before, with two important additions: a PGE₁-induced increasein PDE activity in compartment 1, and a constitutively active PDE incompartment 2. The system is described by the following equations:

$\begin{matrix}{\frac{\mathbb{d}C_{1}}{\mathbb{d}t} = {E_{A\; C} + {\frac{J_{12}}{V_{1}}\left( {C_{2} - C_{1}} \right)} - \frac{A \cdot E_{1} \cdot C_{1}}{K_{M1} + C_{1}}}} & \left( {{Eq}.\mspace{14mu} 1} \right) \\{\frac{\mathbb{d}C_{2}}{\mathbb{d}t} = {{\frac{J_{12}}{V_{2}}\left( {C_{1} - C_{2}} \right)} - \frac{E_{2} \cdot C_{2}}{K_{M2} + C_{2}}}} & \left( {{Eq}.\mspace{14mu} 2} \right) \\{\frac{\mathbb{d}A}{\mathbb{d}t} = {{k_{A}I} - {k_{I}A}}} & \left( {{Eq}.\mspace{14mu} 3} \right)\end{matrix}$where V₁ and V2 are the volumes of compartments 1 and 2, C₁ and C₂ arethe cAMP concentrations, J₁₂ is the flux coefficient betweencompartments, E_(AC) is the synthesis rate of cAMP, E₁ and E₂ are themaximal cAMP hydrolysis rates, K_(M1) and K_(M2) are the Michaelisconstants for PDE activity, A and I are the fraction of active andinactive PDE in compartment 1 (A+I=1), and k_(A) and k_(I) are the rateconstants of PDE activation and inactivation. The parametersJ₁₂=8.0×10⁻¹⁶L/s, V₁=0.040 pL, and V₂=2.0 pL are the same as those usedpreviously (Rich et al., [2000], supra). AC activity is consideredconstant, with E_(AC)=0.13 μM/s. K_(M1), E₁, KM₂, and E₂ are 0.30 μM,0.83 μM/s, 1.0 μM, and 0.0020 μs. The rate constants k_(A) and k₁ are0.0015 s⁻¹ and 0.0010 s⁻¹. The initial and final (300 s) values of A are0.10 and 0.36. The parameters used here reflect similar total PDEactivities near the plasma membrane and throughout the cytosol, in broadagreement with experiments on PDE type IV in several cell types (Houslayet al., Adv. Pharmacol., 44:225–342 [1998]).

Simulations of the model successfully reproduce the local transientchange in cAMP, as well as the rise in total cAMP to a plateau (See,FIG. 5). A PGE₁-induced increase in PDE activity within compartment 1 isrequired to explain the data. Slow efflux of cAMP from the microdomainis ultimately balanced by low rates of hydrolysis within the bulkcytosol. Thus, different relative PDE activities within more than onediffusionally-restricted compartment can explain the generation ofdistinct cAMP signals, even in a simple, nonexcitable cell.

In morphologically-complex cells like neurons, dendritic spines andother subcellular structures have been shown to be diffusionallyisolated compartments (Svoboda et al., Science 272:716–719 [1996];Bridge et al., Science 248:376–378 [1990]; Leblanc and Hume, Science248:372–376 [1990]; Finch and Augustine, Nature 396:753–756 [1998]; andTakechi et al., Nature 396:757–760 [1998]). Surprisingly, resultsobtained during the development of the present invention indicate thatthe concept of three-dimensional barriers to diffusion may begeneralized to simple cells, which are usually considered to contain asingle homogeneous cytosolic compartment. The exact nature of thediffusional barrier is unclear, but it is likely to be formed, at leastin part, by ER membrane that is known to come in close apposition to theplasma membrane (Ma et al., Science 287:1647–1651 [2000]; Akita andKuba, J. Gen. Physiol., 116:697–720 [2000]; and Martin and Fuchs, Proc.Roy. Soc. London B. Biol. Sci., 250:71–76 [1992]). Although localizedCa²⁺ signals have been measured (Roberts et al., J. Neurosci.,10:3664–3684 [1990]; and Jaggar et al., Am. J. Physiol. Cell Physiol.,278:C235–C256 [2000]), these have been attributed primarily to proximityto high-throughput Ca²⁺ sources and the delimiting effects ofhigh-capacity cellular buffers, rather than diffusional barriers.However, it is contemplated that the diffusional barriers to cAMPdescribed herein also influence local Ca²⁺ signals. Regardless, anunderstanding of the mechanism(s) is not necessary in order to use thepresent invention.

Thus, experiments conducted during the development of the presentinvention have resolved distinct cAMP signals in different compartmentsof a simple, nonexcitable cell: a transient signal in microdomains nearthe surface membrane and a signal that rises to a plateau throughout thecell. Diffusional restrictions between the microdomains and the cytosol,as well as differential regulation of PDE activity, are required togenerate such distinct signals. Segregated cAMP signals allow fordifferential regulation of cAMP effector proteins like PKA. This mayexplain a long-standing observation in cardiac myocytes: two agents thatboth trigger rises in cAMP (PGE₁ and isoproterenol) have markedlydifferent downstream effects (Steinberg and Brunton, Ann. Rev.Pharmacol. Toxicol., 41:751–773 [2001]). Similar observations have beenmade in comparing the effects of β₁- and β₂-adrenergic agonists(Steinberg and Brunton, Ann. Rev. Pharmacol. Toxicol., 41:751–773[2001]; Chen-Izu et al., Biophys. J., 79:2547–2556 [2000]; and Davare etal., Science 293:98–101 [2001]). In this context, it is interesting toreconsider the possible functions of A-kinase anchoring proteins(AKAPs), the scaffolds that tether PKA to cellular targets (Felicielloet al., J. Mol. Biol., 308:99–114 [2001]; and Gray et al., Curr. Opin.Neurobiol., 8:330–334 [1998]). In addition to creating two-dimensionalprotein arrays that help to ensure the phosphorylation of certainproteins, AKAPs are likely to direct PKA to diffusionally isolatedcellular regions in which distinct cAMP signals are produced. Thetransient nature of the signals measured here limits the diffusionalspread of cAMP. However, these signals should still allow for aprolonged activation of PKA because the reassociation of PKA subunits isslow (Ogried and Doskeland, Biochem., 22:1686–1696 [1983]; andHarootunian et al., Mol. Biol. Cell 4:993–1002 [1993]).

Improving the cAMP-Sensing Capabilities of CNG Channels

During the development of the present invention, experiments indicatedthat adenovirus-expressed rat olfactory cyclic CNG channels have severalproperties that make them excellent cAMP sensors. These propertiesinclude their location at the plasma membrane, rapid gating kinetics,and lack of desensitization. Furthermore, these channels appear toco-localize with AC in discrete regions of the membrane, which allowsthe measurement of localized cAMP signals. However, there are severallimitations to the use of WT CNG channels to detect changes in cAMP.First, WT channels have a low apparent affinity for cAMP (Table 1, belowand FIG. 6, Panel A), which makes it difficult to detect the low cAMPconcentrations that activate protein kinase A (PKA). Second, thesechannels are activated more readily by cGMP than cAMP (See, FIG. 6,Panel A; See also, Dhallan et al., Nature 347:184–187 [1990]). Third,they can also be activated directly by nitric oxide (NO; Broillet, J.Biol. Chem., 275:15135–15141 [2000]). Fourth, the binding of theCa²⁺-CaM complex to these channels strongly inhibits channel opening(Liu et al., Science 266:1348–1354 [1994]). To overcome theselimitations, the properties of the WT channel were modified, so as toprovide the improvements of the present invention.

First, the mutation E583M was introduced into the rat olfactory CNGchannel α subunit (See, FIG. 1 and SEQ ID NO:1). Vamum et al. (Varnum etal, Neuron 15:619–625 [1995]) showed that mutation of the correspondingresidue in the alpha subunit of the bovine retinal rod CNG channel(D604M) increased the sensitivity to cAMP and decreased the sensitivityto cGMP. As the olfactory channel has a higher overall sensitivity tocyclic nucleotides than the rod channel, the results of theseexperiments were not predictable. However, in excised patches, the E583Mchannel displayed increased sensitivity for cAMP and decreasedsensitivity for cGMP (Table 1; compare FIG. 6, Panels A and B).Furthermore, cGMP was only a partial agonist of the E583M channel. Thecurrent elicited by saturating cGMP was ˜40% of the current elicited bysaturating cAMP (See, Table 1, and FIG. 6, Panel B).

To further increase the sensitivity to cAMP, a novel second mutant,C460W/E583M, was constructed (See, FIG. 2 and SEQ ID NO:2). As indicatedin Table 1, and FIG. 6, Panel C, the double mutant was considerably moresensitive to cAMP than the E583M channel (˜10-fold lower K_(1/2)).

TABLE 1 Characteristics of CNG Channels Used as cAMP Sensors MembranePotential K_(1/2) ^(cAMP) K_(1/2) ^(cGMP) I_(max) ^(cGMP)/ Channel (mV)(μM) N^(cAMP) (μM) N^(cGMP) I_(max) ^(cAMP) WT +50 36 ± 5  2.2 ± 0.1 1.6± 0.1 2.3 ± 0.1 1.0 E583M 10.5 ± 0.2  2.4 ± 0.2 28 ±  2  2.4 ± 0.12 0.40± 0.10 C460W/ 1.2 ± 0.3 2.7 ± 0.2 12 ± 2  2.8 ± 0.3 0.84 ± 0.10 E583MΔ61-90/ 14.5 ± 1.8  2.1 ± 0.1 36 ± 4  1.8 ± 0.2 0.16 ± 0.08 C460W/ E583MWT −50 36 ± 5  2.2 ± 0.1 1.3 ± 0.4 2.5 ± 0.4 1.0 E583M 9 ± 2 2.2 ± 0.132 ± 4  1.9 ± 0.2 0.35 ± 0.16 C460W/ 0.89 ± 0.23 2.2 ± 0.3 6.2 ± 1   2.7± 0.1 0.50 ± 0.10 E583M Δ61-90/ 10.5 ± 0.1  2.2 ± 0.1 16 ± 1  2.6 ± 0.20.09 ± 0.06 C460W/ E583M Data are presented as mean ± SEM of threeexperiments. Hill equation parameters are defined herein. I_(max)^(cGMP) /I_(max) ^(cAMP) is the current induced by saturating cGMPdivided by the current induced by saturating cAMP.

To remove regulation of the channel by Ca²⁺-CaM binding residues 61through 90 were deleted using method previously described (Liu et al.,Science 266:1348–1354 [1994]). This channel, ΔA61-90/C460W/E583M (See,FIG. 3 and SEQ ID NO:3), is almost as sensitive to cAMP as the E583Mchannel (See, Table 1, and FIG. 6, Panel D), yet it is virtuallyinsensitive to cGMP. In fact, the current elicited by saturating cGMPwas <20% of the current elicited by saturating cAMP (See, Table 1, andFIG. 6, Panel D). Thus, two very useful channel constructs weregenerated during the development of the present invention for themeasurement of cAMP, namely the ΔA61-90/C460W/E583M channel that issensitive to cAMP at the upper end of the physiological range (˜1 to 50μM); and the C460W/E583M channel that is sensitive to cAMP at the lowerend of the physiological range (˜0.1 to 5 μM).

In sum, the modified CNG channels of the present invention provide thefirst ion channels that have been tailored for the measurement of cAMP,as they are primarily activated by cAMP. Although other mutations havebeen previously made at C460, the present invention provides mutationsthat exhibit greater changes in cAMP sensitivity. Furthermore, mutationsat these positions have apparently never been combined. As little isknown about the three-dimensional structure of CNG channels, there is noway to predict the effects of the combined mutations. In addition, thedeletion of residues 61–90, which results in the removal of Ca²⁺-CaMsensitivity, dramatically lowers cyclic nucleotide sensitivity (i.e.,10- to 20-fold). Thus, the prior art teaches away from the mutations ofthe present invention and their use in assay systems with increasedsensitivity for cAMP measurements.

Assessment of the Relative cAMP Sensitivity of Channel Constructs InVivo

Next, experiments were conducted to assess the ability of each channelconstruct to detect increases in local cAMP concentration. Changes incAMP concentration were detected using fura-2 to monitor Ca²⁺ influxthrough CNG channels, as described in the Examples. Ca²⁺ influx inducedby different concentrations of forskolin, an AC activator, in thepresence of 100 μM IBMX, a nonselective PDE inhibitor was measured. Thisapproach allowed comparisons to be made between the cAMP sensitivitiesof the different channels to changes in cAMP, regardless of variationsin expression levels between experiments. In HEK-293 cells expressingthe WT channel, addition of forskolin was followed by a brief delay andan increase in Ca²⁺ influx (See, FIG. 7, Panel A). The delay decreasedand the rate of Ca²⁺ influx increased in a dose-dependent manner.Neither effect was saturated at 50 μM forskolin. In cells expressing theE583M channel, addition of forskolin was also followed by a brief delayand an increase in Ca²⁺ influx (FIG. 7, Panel B). As with the WTchannel, the delay and the initial rate of Ca²⁺ influx weredose-dependent. However, both effects saturated at 20 μM forskolin, asthe 20 and 50 μM forskolin traces overlap, as indicated in FIG. 7, PanelB. In cells expressing the C460W/E583M channel, both the delay and therate of Ca²⁺ influx were saturated at 5 μM forskolin (FIG. 7, Panel C).In cells expressing the Δ61-90/C460W/E583M channel, addition offorskolin caused similar increases in Ca²⁺ influx as the E583M channel,including saturation of the delay and rate of influx at 20 μM (compareFIG. 7, Panel B with FIG. 7, Panel D). The increased response of themodified channels to forskolin treatment is consistent with the apparentcAMP affinities measured in excised patches in the absence of Ca²⁺-CaM(C460W/E583M>E583M˜Δ61-90/C460W/E583>WT channels). There are severalfactors that may have contributed to the apparent lack of a Ca²⁺-CaMeffect on the channels. As indicated in the Examples, the concentrationsof fura-2 being used are likely overwhelming high-affinity cellular Ca²⁺buffers, in terms of the fraction of incoming Ca²⁺ that is bound, andtherefore fura-2 is probably significantly reducing the effects ofCa²⁺-CaM. It is also contemplated that other possibilities include:HEK-293 cells do not produce enough CaM to regulate heterologouslyexpressed CNG channels, or that the CaM concentration withinmicrodomains is too low to significantly regulate the channels. Verysmall forskolin-induced changes in Ca²⁺ influx were observed in HEK-293cells not expressing CNG channel constructs (See, FIG. 8).

PDE Activity in HEK-293 Cells

As discussed herein, CNG channels monitor cAMP produced in subcellularcompartments near the plasma membrane. To assess the extent to which PDEactivity affects cAMP levels, forskolin-induced Ca²⁺ influx in thepresence and absence of PDE inhibitors was measured. Initially theeffects of the nonselective PDE inhibitor, IBMX, were examined usingeach CNG channel construct as a cAMP sensor. In cells expressing the WTchannel, there was little or no change in Ca²⁺ influx in response to anintermediate forskolin concentration (10 μM; See, FIG. 9, Panel A).After a three minute pretreatment with 100 μM IBMX only a modestforskolin-induced increase in Ca²⁺ influx was observed (See, FIG. 9,Panel A), arising from Ca²⁺ entry through WT channels. In cellsexpressing the E583M channel, a moderate forskolin-induced increase inCa²⁺ influx was observed in the absence of PDE inhibitors (See, FIG. 9,Panel B). After pretreatment with IBMX, forskolin caused a robustincrease in Ca²⁺ influx. In cells expressing the C460W/E583M channel,forskolin-induced increases in Ca²⁺ influx were readily observable, evenin the absence of PDE inhibitors (See, FIG. 9, Panel C). Thus, asindicated by a comparison with FIG. 9, Panels B and D, this channel iscapable of detecting cAMP in cells with low AC activity. Using the61-90/C460W/E583M channel as a sensor gave similar forskolin-inducedchanges in Ca²⁺ influx as the E583M channel, both in the absence andpresence of IBMX. These results demonstrate that, in HEK-293 cells,basal PDE activity limits the accumulation of cAMP following a moderatestimulus. However, even in the absence of PDE inhibitors, followingstimulation of AC with high concentrations of forskolin (50–100 μM),cAMP reaches levels high enough to activate WT CNG channels (data notshown).

Next, experiments were conducted to pharmacologically identify the PDEtype(s) that regulates cAMP levels near CNG channels. The C460W/E583Mchannels were used to monitor forskolin-stimulated cAMP accumulation inthe presence and absence of a series of PDE inhibitors. The reportedIC₅₀ values of inhibitors used in this study for each PDE type are givenin Table 3. The PDE inhibitor concentrations used were typically atleast 5-fold higher than the most potent IC₅₀. Either vehicle or PDEinhibitors were added at 0 s and 1 μM forskolin was added at 180 s (See,FIG. 10). As indicated in this Figure, in the absence of PDE inhibitorslittle or no forskolin-induced Ca²⁺ influx was observed; whereas, in thepresence of IBMX (10 or 100 μM), or the PDE-type-IV specific inhibitorRO-20-1724 (10 μM) significant forskolin-induced Ca²⁺ influx wasobserved (See, FIG. 10, Panels A, B and G). Two other PDE type IVinhibitors significantly increased the forskolin-induced Ca²⁺ influx,rolipram (FIG. 11, Panel C) and etazolate (not shown). Inhibitorsspecific to other PDE families did not affect forskolin-induced Ca²⁺influx. Similar results were obtained in HEK-293 cells expressing eitherthe WT or ΔA61-90/C460W/E583M channels by stimulating cAMP productionwith higher forskolin concentrations (10 μM, not shown), or whenprostaglandin E₁ (1 μM) was used to stimulate AC activity (not shown).In these experiments, inhibitor concentrations were used at whichspecific PDE types should be inhibited. However, many PDE inhibitors arenot completely specific (See, Table 3). Also, PDE types VIII and IX areinsensitive to IBMX as well as most PDE inhibitors (See, referenceslisted for Table 3). Unfortunately, dipyridamole, the inhibitor to whichthey are most sensitive, fluoresces and, as such, cannot be used in thisassay.

In Vivo Estimates of PDE Inhibitor K_(I)

To further establish that PDE type IV is responsible for the observedIBMX-sensitive PDE activity, the K_(I)'s (inhibition constants) of IBMXand two PDE type IV inhibitors (RO-20-1724 and rolipram) were estimatedin vivo. This required developing a quantitative framework to assess therelationship between cAMP synthesis, hydrolysis, and redistributionthroughout the cell. Thus, the following formalism was adopted:

$\begin{matrix}{\frac{\mathbb{d}\lbrack{cAMP}\rbrack}{\mathbb{d}t} = {C - \frac{V_{\max} \cdot \lbrack{cAMP}\rbrack}{{K_{m} \cdot \left( {1 + \frac{\lbrack I\rbrack}{K_{I}}} \right)} + \lbrack{cAMP}\rbrack} - {k_{f} \cdot \lbrack{cAMP}\rbrack}}} & \left( {{Eq}.\mspace{14mu} 4} \right)\end{matrix}$where C is the steady-state rate of cAMP synthesis by AC, V_(max) is themaximal rate of cAMP hydrolysis, K_(m) is the Michaelis constant forPDE, and k_(f) is the rate constant of cAMP flux out of the microdomain.In order to estimate K_(I) for the PDE inhibitors two assumptions weremade. First, it was assumed that at low levels of AC stimulation and PDEinhibition, the concentration of local cAMP is low and diffusion of cAMPout of the microdomain is negligible. Second, the assumption was madethat cAMP levels reach steady-state shortly after AC stimulation (i.e.,equal rates of synthesis and hydrolysis). With these assumptions, Eq. 4can be simplified to:

$\begin{matrix}{\lbrack{cAMP}\rbrack = {\frac{C \cdot K_{m}}{V_{\max} - C} \cdot \left( {1 + \frac{\lbrack I\rbrack}{K_{I}}} \right)}} & \left( {{Eq}.\mspace{11mu} 5} \right)\end{matrix}$Interestingly, this equation reveals that when the inhibitorconcentration is equal to K_(I) the cAMP concentration is twice that inthe absence of inhibitor. At low cAMP concentrations (<K_(1/2) for thechannel), the cAMP concentration is proportional to the square root ofthe Ca²⁺ influx rate (the Hill coefficient for channel activation isapproximately 2). Thus, K_(I) can be estimated using a linear fit to thesquare root of the slopes of the fluorescence traces as a function ofinhibitor concentration (See, FIG. 11).

The high cAMP affinity C460W/E583M channels were used to detect changesin cAMP concentration following pretreatment with PDE inhibitors (addedat 0 s) and modest forskolin stimulation (0.5 μM added at 180 s). IBMX,RO-20-1724, and rolipram were completely equilibrated across the plasmamembrane of HEK-293 cells in <180 s (data not shown). The assumptionthat cAMP levels reach steady-state is supported by the long-lastinglinear rise in Ca²⁺ concentration following forskolin stimulation (asteady Ca²⁺ influx rate reflects a constant cAMP level; See, FIG. 12).Dose response relations for the three PDE inhibitors are shown in FIG.11. Based upon fits to this data with Eq. 5 (See, insets in FIG. 11,Panels A–C), the K_(I) values were estimated to be 11±2 μM (IBMX),0.13±0.02 μM (RO-20-1724), and 0.07±0.02 μM (rolipram), n=4, which areconsistent with published IC₅₀ values from in vitro experiments (See,Table 3). To ensure that the results were independent of channelconstruct, the K_(I) for RO-20-1724 using the lower cAMP affinityΔ61-90/C460W/E583M channels was also estimated. The K_(I) (0.15±0.02 μM,n=3) was indistinguishable from that estimated using C460W/E583Mchannels. These data strongly indicate that PDE type IV is primarilyresponsible for the IBMX-sensitive component of PDE activity monitoredusing CNG channels and, thus, for regulating localized cAMPconcentration in HEK-293 cells.

At higher PDE inhibitor concentrations, the square root of Ca²⁺ influxrate, which is proportional to cAMP concentration, deviates fromlinearity (See, FIG. 11). It is contemplated that at higher inhibitorconcentrations, cAMP may reach levels≧K_(1/2) of the channel. At theseconcentrations, the relationship between Ca²⁺ influx rate and cAMPconcentration deviates from a simple square law. This does not explainwhy at high levels of PDE inhibition the Ca²⁺ influx rate reaches aplateau. When PDE activity is completely inhibited, forskolin-stimulatedcAMP accumulation would continually increase in a confined region offree diffusion. This, in turn, would lead to higher Ca²⁺ influx rates.To ensure that the plateau was not primarily due to channel saturationor Ca²⁺ homeostatic mechanisms, Ca²⁺ influx rates induced by 50 μMforskolin and maximal PDE inhibition were measured in the sameexperiment. Under these conditions, the Ca²⁺ influx rate greatlyexceeded the influx rates induced by 0.5 μM forskolin with PDEinhibition. At increased cAMP concentrations, cAMP efflux from themicrodomain is expected to increase; this increased efflux could createthe observed plateau. This would be consistent with the diffusionallyrestricted microdomain model (See, Rich et al. [2000], supra).

PDE Activity in GH4C1 Cells

To further test the utility of this approach, PDE activity in excitableGH4C1 pituitary cells was examined. In all of the experiments shown(See, FIGS. 13–14, and Table 2), 1 μM nimodipine was added at time zeroto block Ca²⁺ influx though voltage-gated Ca²⁺ channels (triggered bymembrane depolarization due to Ca²⁺ and Na⁺ influx through CNGchannels). This concentration of nimodipine was sufficient to block Ca²⁺influx through voltage-gated Ca²⁺ channels activated by membranedepolarization in 24 mM external KCl, and did not alter forskolin orpCPT-cGMP induced Ca²⁺ influx through CNG channels expressed in HEK-293cells (data not shown). FIGS. 13 and 14 each depict experiments done ona single batch of cells. As before, similar results were obtained onthree other batches. In the experiments that produced the data shown inTable 2, cAMP concentrations were monitored using Ca²⁺ influx throughC460W/E583M channels. In these experiments, cells expressing WT CNGchannels, as well as control cells (i.e., cells that did not express CNGchannels) were used. Either 100 μM IBMX or vehicle (−IBMX; control) wereadded at time zero. Then, 50 μM forskolin were added at 180 seconds.d(ΔF/F₀)/dt, which is proportional to the Ca²⁺ influx rate, wasestimated as described in the Examples. Data are provided asmean±standard error. IBMX or vehicle was added at 60 seconds (firstarrow in FIG. 13). Either 10 μM forskolin or 100 nM VIP were added at240 seconds (second arrow in FIG. 13). In the absence of IBMX, largeforskolin or VIP-induced increases in Ca²⁺ influx were observed. Whenthe local PDE activity was inhibited by 100 μM IBMX, substantial basalactivity was revealed. This level of basal AC activity was quitedifferent from that observed in HEK-293 cells (See, FIGS. 9 and 10). 1μM nimodipine was added at time 0 to block endogenous voltage-gated Ca²⁺channels.

TABLE 2 Effect of PDE Inhibitors on Basal cAMP Levels in GH4C1 CellsInhibitor (μM) d(ΔF/F₀)/dt No. of Experiments 100 IBMX s⁻¹ 5 1008-methoxymethyl-IBMX 0.0025 ± 0.0009 4  10 EHNA 0.0029 ± 0.0011 3  1Trequinsin 0 3  15 Quazinone 0 3 100 RO-20-1724 0 4 100 Rolipram 0 4  50Zaprinast 0 3

FIG. 13, Panel A shows the forskolin-induced responses of cellsexpressing WT channels. Either vehicle or 100 μM IBMX was added at 0 s,and 50 μM forskolin was added at 180 s. After the addition of forskolin,there was a short delay followed by an increased Ca²⁺ influx. The slopeof the Ca²⁺ influx was greater in the presence of IBMX, indicatinghigher cAMP levels. In control cells (i.e., cells not expressing CNGchannel constructs), no forskolin-induced Ca²⁺ influx was observed ineither the presence or absence of IBMX (See, FIG. 13, Panel B). This wastrue of controls done for all experimental protocols.

Next, forskolin-induced responses and the effects of IBMX in cellsexpressing the high cAMP affinity construct, C460W/E583M were examined.In these experiments either vehicle or 100 μM IBMX was added at 60 s(FIG. 13, Panels C, D). Interestingly, the addition of IBMX triggeredCa²⁺ influx that was not observed in either control cells or cellsexpressing the WT channel. Subsequent addition of 10 μM forskolin (See,FIG. 13, Panel C) or 100 nM vasoactive intestinal peptide (VIP; FIG. 13,Panel D) caused additional cAMP accumulation and Ca²⁺ influx. Acomparison of responses measured with the WT and C460W/E583M channelsindicates that the IBMX-induced response was not due to an increase inlocal cGMP concentration. Moreover, 100 μM IBMX did not alter CNGchannel activity monitored in excised patches (data not shown). Thus,these data indicate that the IBMX-induced Ca²⁺ influx was due primarilyto an increase in cAMP arising from basal AC activity.

To identify the PDE family or families that regulate the local cAMPconcentration in GH4C1 cells, cAMP accumulation was monitored in thepresence and absence of PDE inhibitors (See, Table 2). Again, the highcAMP affinity C460W/E583M channels were used. PDE inhibitors were addedat 60 s. Although the addition of IBMX alone triggered Ca²⁺ influx, 10μM forskolin were added at 240 s as a positive control for CNG channelactivity. Only the nonspecific PDE inhibitor, IBMX, and the PDE-type-Ispecific inhibitor, 8-methoxymethyl-IBMX, induced Ca²⁺ influx throughCNG channels (Table 2). This PDE is likely to have a low K_(m) for cAMPbecause it is capable of regulating cAMP at concentrations too low forthe WT or Δ61-90/C460W/E583M channels to detect. Thus, it iscontemplated that a Ca²⁺-CaM stimulated PDE (type I) is primarilyresponsible for controlling basal cAMP signals in GH4C1 cells.

In addition, PDE activity was monitored using ΔA61-90/C460W/E583Mchannels to detect changes in cAMP concentration (See, FIG. 14). PDEinhibitors and forskolin were added as described above. With thisconstruct, it was not possible to observe changes in cAMP concentrationdue to inhibition of PDE activity alone. However, PDE inhibitors causedan increase in forskolin-induced Ca²⁺ influx. As observed using the highcAMP affinity C460W/E583M channels, both IBMX and the PDE-type-Ispecific inhibitor 8-methoxymethyl-IBMX significantly inhibited PDEactivity (See, FIG. 14, Panels A and B). Interestingly, the PDE-type-IVspecific inhibitors, RO-20-1724 and rolipram, also inhibited PDEactivity (See, FIG. 14, Panels C and D). Inhibitors specific to otherPDE types had no effect on the forskolin-induced Ca²⁺ influx (data notshown). No RO-20-1724- or rolipram-sensitive PDE was observed using thehigh cAMP affinity channel (See, Table 2). It is contemplated that Ca²⁺influx through the C460W/E583M channels was saturated at cAMPconcentrations below the K_(m) for this PDE. Thus, it is contemplatedthat there are two different PDE types that regulate local cAMP signalsin GH4C1 cells: a low K_(m), 8-methoxymethyl-IBMX sensitive PDE (typeI); and a high K_(m), RO-20-1724-, rolipram-sensitive PDE (type IV).

The methods described herein are suitable for determination of whetheran unknown compound modulates PDE activity or adenylyl cyclase activity.Known inhibitors of PDE (either type-specific inhibitors described aboveor non-type-specific inhibitors such as IBMX) are mixed, at saturatingconcentrations, with the unknown compound. If the known PDE inhibitors(particularly type-specific ones) block the effect of the unknowncompound (e.g., a rise in cAMP detected as described herein), then theunknown compound is categorized as a candidate PDE modulator. If aseries of PDE inhibitors do not prevent the effect of the unknowncompound, then the compound is categorized as a candidate adenylylcyclase modulator. These initial determinations are then verified byusing more difficult and time-consuming assays with purified proteins.Thus, the methods described herein provide very useful initial screeningtools for compounds that are capable of modulating PDE or adenylylcyclase activity in living cells.

Definitions

As used herein, the terms “patch clamp” and “patch clamp recordingmethods” refer to methods that involve sealing the tip of a small (e.g.,about 1 micron) orifice glass pipette to the membrane of a cell. Underoptimal conditions, a seal resistance of greater than one billion ohmsis formed around the rim of the pipette tip between the cell membraneand the glass. When the pipette is connected to an appropriateamplifier, small currents across the patch of membrane inside thepipette tip can be recorded. This initial configuration is referred toas a “cell-attached patch.” When such a patch is pulled from the cell,an “excised inside-out patch” forms, with the cytoplasmic face of thepatch membrane facing the bathing solution. Alternatively, in thecell-attached configuration, the membrane inside the patch pipette canbe ruptured with light suction to provide access to the cell cytoplasm.In this condition, currents are recorded from the entire cell (i.e.,“whole-cell configuration”). In the “perforated patch configuration,” apore-forming antimicrobial (e.g., nystatin) is added to the pipettesolution to gain electrical access to the cell's interior. This allowscurrents to be recorded from the entire cell, while retaining divalentcations and larger molecules (e.g., cAMP) in the cell.

As used herein, the term “adenoviruses” (Ad) refers to thedouble-stranded DNA viruses of the Adenoviridae. The genome ofadenoviruses (˜36 kb) is complex and contains over 50 open readingframes (ORFs). These ORFs are overlapping and genes encoding one proteinare often embedded within genes coding for other Ad proteins. Expressionof Ad genes is divided into an early and a late phase. Early genes arethose transcribed prior to replication of the genome while late genesare transcribed after replication. The early genes comprise E1a, E1b,E2a, E2b, E3 and E4. The E1a gene products are involved intranscriptional regulation, while the E1b gene products are involved inthe shut-off of host cell functions and mRNA transport. E2a encodes thea DNA-binding protein (DBP), while E2b encodes the viral DNA polymeraseand preterminal protein (pTP). The E3 gene products are not essentialfor viral growth in cell culture. The E4 region encodes regulatoryprotein involved in transcriptional and post-transcriptional regulationof viral gene expression, and subset of the E4 proteins are essentialfor viral growth. The products of the late genes (e.g., L1–5) arepredominantly components of the virion as well as proteins involved inthe assembly of virions. The VA genes produce VA RNAs which block thehost cell from shutting down viral protein synthesis.

Adenoviruses or recombinant Ad vectors have been exploited for thedelivery of foreign genes to cells for a number of reasons including thefact that Ad vectors have been shown to be highly effective for thetransfer of genes into a wide variety of tissues in vivo and the factthat Ad infects both dividing and non-dividing cells.

As used herein, the term “virus” refers to obligate, ultra microscopic,intracellular parasites incapable of autonomous replication (i.e.,replication requires the use of the host cell's machinery).Adenoviruses, as noted above, are double-stranded DNA viruses. The leftand right inverted terminal repeats (ITRs) are short elements located atthe 5′ and 3′ termini of the linear Ad genome, respectively and arerequired for replication of the viral DNA. The two ITRs are invertedrepeats of each other. The “adenovirus packaging sequence” refers to thesequence which comprises five (AI–AV) Packaging signals and is requiredfor encapsulation of the mature linear genome; the packaging signals arelocated from ˜194 to 358 bp in the Ad genome (about 0.5–1.0 mu).

As used herein, the term “primary cell” refers to a cell which isdirectly obtained from a tissue or organ of an animal whether or not thecell is in culture.

As used herein, the term “cultured cell” refers a cell which has beenmaintained and/or propagated in vitro. Cultured cells include primarycultured cells and cell lines.

As used herein, the term “primary cultured cells” refers to primarycells which are cultured in vitro and which preferably, though notnecessarily, are capable of undergoing ten or fewer passages in in vitroculture before senescence and/or cessation of proliferation.

As used herein, the terms “cell line” and “immortalized cell” refer to acell which is capable of a greater number of cell divisions in vitrobefore cessation of proliferation and/or senescence as compared to aprimary cell from the same source. A cell line includes, but does notrequire, that the cells be capable of an infinite number of celldivisions in culture. The number of cell divisions may be determined bythe number of times a cell population may be passaged (i.e.,subcultured) in in vitro culture. Passaging of cells is accomplished bymethods known in the art. Cell lines may be generated spontaneously orby transformation. A “spontaneous cell line” is a cell line which arisesduring routine culture of cells. A “transformed cell line” refers to acell line which is generated by the introduction of a “transgene”comprising nucleic acid (usually DNA) into a primary cell or into afinite cell line by means of human intervention

Cell lines include, but are not limited to, finite cell lines andcontinuous cell lines. As used herein, the term “finite cell line”refers to a cell line which is capable of a limited number (from about 1to about 50, more preferably from about 1 to about 40, and mostpreferably from about 1 to about 20) of cell divisions prior tosenescence. The term “continuous cell line” refer to a cell line whichis capable of more than about 50 (and more preferably, an infinitenumber of) cell divisions. A continuous cell line generally, althoughnot necessarily, also has the general characteristics of a reduced cellsize, higher growth rate, higher cloning efficiency, increasedtumorigenicity, and/or a variable chromosomal complement as compared tothe finite cell line or primary cultured cells from which it is derived.

The term “transgene” as used herein refers to any nucleic acid sequencewhich is introduced into the cell by experimental manipulations. Atransgene may be an “endogenous DNA sequence” or a “heterologous DNAsequence” (i.e., “foreign DNA”). The term “endogenous DNA sequence”refers to a nucleotide sequence which is naturally found in the cellinto which it is introduced so long as it does not contain somemodification (e.g., a point mutation, the presence of a selectablemarker gene, etc.) relative to the naturally-occurring sequence. Theterm “heterologous DNA sequence” refers to a nucleotide sequence whichis ligated to, or is manipulated to become ligated to, a nucleic acidsequence to which it is not ligated in nature, or to which it is ligatedat a different location in nature. Heterologous DNA is not endogenous tothe cell into which it is introduced, but has been obtained from anothercell. Heterologous DNA also includes an endogenous DNA sequence whichcontains some modification. Generally, although not necessarily,heterologous DNA encodes RNA and proteins that are not normally producedby the cell into which it is expressed. Examples of heterologous DNAinclude reporter genes, transcriptional and translational regulatorysequences, selectable marker proteins (e.g., proteins which confer drugresistance), etc.

The term “label” as used herein refers to any atom or molecule which canbe used to provide a detectable (preferably quantifiable) signal, andwhich can be attached to a nucleic acid or protein. Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like.

As used herein, the term “detection signal” refers to an indicator ofthe presence or absence of a compound of interest. For example, in somemethods of the present invention, fluorescence (i.e., the detectionsignal) is measured at particular excitation and emission wavelengths,to detect Ca²⁺ influx. In these methods, Ca²⁺ influx was found to causea decrease in fluorescence (ΔF), which was expressed relative to thepre-stimulus fluorescence (F₀) to correct for variations in dyeconcentration, and to allow for comparison of results on differentbatches of cells. Although fluorescence was used in the Examplesdescribed herein, it is not intended that the present invention belimited to systems that rely upon use of a fluorescent signal. Indeed,assay systems utilizing other signals find use in the present invention,including but not limited to chemiluminescence, bioluminescence, etc.,as known in the art.

As used herein, the term “second messenger” refers to small molecules orions that are generated in the cytoplasm of cells in response to bindingof a signal molecule to its receptor on the outer surface of the cellmembrane. Two major classes of second messengers are known, includingone in which cAMP is involved and one which involves a combination ofcalcium ions and either inositol triphosphate or diacylglycerol.

As used herein, the term “ion channel” refers to a pathway (i.e., achannel) through the cell membrane, which allows ions to enter and/orexit cells.

As used herein, the term “gated ion channel” refers to ion channels thatexhibit selectivity in the timing and/or properties of ions that areallowed to pass through an ion channel and enter or exit a cell.Typically, these channels have a structure which determines theparticles that are allowed to enter and/or exit the cell.

The terms “functional calcium channel” and “biologically active calciumchannel” interchangeably refer to a calcium channel which allows entryinto a cell of a calcium ion in response to a stimulus. Such entry maybe determined by measuring the amount of current which flows through thecalcium channel in response to the stimulus. Alternatively, a functionalcalcium channel refers to a calcium channel which binds ligands thathave affinity for a calcium channel. For ligand binding assays of arecombinant calcium channel, it is preferred that the host cell which isused for testing the function of the recombinant calcium channel notproduce endogenous calcium channel subunits that are of a type or in anamount that interferes with the detection of the recombinant calciumchannel. Methods for determining the function of a calcium channel areknown in the art.

The term “compound that modulates calcium channel activity” andgrammatical equivalents thereof refers to a compound that alters (i.e.,reduces or increases) the ability of a calcium channel to pass calciumions as measured by, for example, the current flowing through thecalcium channel. Such compounds include, but are not limited to, calciumchannel agonists (e.g., Goldin et al., U.S. Pat. No. 5,312,928, hereinincorporated by reference) and antagonists, and compounds that exerttheir effect on the activity of the calcium channel directly orindirectly.

As used herein, the term “permeant” refers to molecules which arecapable of entering cells by means of ion channels or other mechanisms.“Permeant” includes, but is not limited to ions such as chloride,potassium, sodium, and thiocyanate.

The term “biologically active” as used herein, refers to a protein orother biologically active molecules (e.g., catalytic RNA) havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, immunologically active refers to thecapability of the natural, recombinant, or synthetic human protein, orany oligopeptide or polynucleotide thereof, to induce a specific immuneresponse in appropriate animals or cells and to bind with specificantibodies.

As used herein, the term “agonist” refers to molecules or compoundswhich mimic the action of a “native” or “natural” compound. Agonists maybe homologous to these natural compounds in respect to conformation,charge or other characteristics. Thus, agonists may be recognized byreceptors expressed on cell surfaces. This recognition may result inphysiologic and/or biochemical changes within the cell, such that thecell reacts to the presence of the agonist in the same manner as if thenatural compound was present. Agonists may include proteins, nucleicacids, carbohydrates, or any other molecules which bind or interact withgated ion channels.

As used herein, the terms “antagonist” and “inhibitor” refer tomolecules or compounds which inhibit the action of a “native” or“natural” compound. Antagonists may or may not be homologous to thesenatural compounds in respect to conformation, charge or othercharacteristics. Thus, antagonists may be recognized by the same ordifferent receptors that are recognized by an agonist. Antagonists mayhave allosteric effects which prevent the action of an agonist (e.g.,prevent opening of the chloride ion channel). Or, antagonists mayprevent the function of the agonist (e.g., by blocking the passage ofchloride ions in the channels). In contrast to the agonists,antagonistic compounds do not result in physiologic and/or biochemicalchanges within the cell such that the cell reacts to the presence of theantagonist in the same manner as if the natural compound was present.Antagonists and inhibitors may include proteins, nucleic acids,carbohydrates, or any other molecules which bind or interact with gatedion channels. As used herein, the term modulate, refers to a change oran alteration in the biological activity. Modulation may be an increaseor a decrease in protein activity, a change in binding characteristics,a change in ion passage through a gated channel, or any other change inthe biological, functional, or immunological properties associated withthe activity of a protein or other structure of interest.

As used herein, the term “transformation” refers to the introduction offoreign genetic material into a cell or organism. Transformation may beaccomplished by any method known in the art which permits the successfulintroduction of nucleic acids into cells and which results in theexpression of the introduced nucleic acid. For example, transformationmay be used to introduce cloned DNA encoding a normal or mutant cyclicnucleotide gated ion channel into a cell which normally does not expressthis ion channel. Transformation may be accomplished through use of anyexpression vector. For example, the use of baculovirus to introduceforeign nucleic acid into insect cells is contemplated. The term“transformation” also includes methods such as P-element mediatedgermline transformation of whole insects. As used herein, the term“transformation” also includes but is not limited to methods such as“transfection” and “transduction.”

The term “transfection” generally refers to the introduction of foreignDNA into eukaryotic cells, but may also be used to refer to theintroduction of foreign DNA into prokaryotic cells. Transfection may beaccomplished by a variety of means known to the art including but notlimited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediatedtransfection, polybrene-mediated transfection, electroporation,microinjection, liposome fusion, lipofection, protoplast fusion, viralinfection (transduction), and biolistics.

As used herein, the term “gene” refers to the deoxyribonucleotidesequences comprising the coding region of a structural gene andincluding sequences located adjacent to the coding region on both the 5′and 3′ ends for a distance of several kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. A genomicform or clone of a gene contains coding sequences, termed exons,alternating with non-coding sequences termed “introns” or “interveningregions” or “intervening sequences.” Introns are segments of a genewhich are transcribed into heterogenous nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. In addition tocontaining introns, genomic forms of a gene may also include sequenceslocated on both the 5′ and 3′ end of the sequences which are present onthe RNA transcript. These sequences are referred to as “flanking”sequences or regions (these flanking sequences are located 5′ or 3′ tothe non-translated sequences present on the mRNA transcript). The 5′flanking region may contain regulatory sequences such as promoters andenhancers which control or influence the transcription of the gene. The3′ flanking region may contain sequences which direct the termination oftranscription, posttranscriptional cleavage and polyadenylation.

As used herein, the term “coding region,” when used in reference to astructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof an mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

As used herein, the term “structural gene” refers to a DNA sequencecoding for RNA or a protein. In contrast, “regulatory genes” arestructural genes which encode products (e.g., transcription factors)which control the expression of other genes.

As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, enhancer elements, etc. Promoters andenhancers consist of short arrays of DNA sequences that interactspecifically with cellular proteins involved in transcription (Maniatiset al., Science 236:1237 [1987]). Promoter and enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inyeast, insect and mammalian cells and viruses (analogous controlelements [i.e., promoters], are also found in prokaryotes). Theselection of a particular promoter and enhancer depends on what celltype is to be used to express the protein of interest. Some eukaryoticpromoters and enhancers have a broad host range while others arefunctional in a limited subset of cell types (for review see Voss, etal., Trends Biochem. Sci., 11:287 [1986]; and Maniatis, et al., Science236:1237 [1987]). For example, the SV40 early gene enhancer is veryactive in a wide variety of cell types from many mammalian species andhas been widely used for the expression of proteins in mammalian cells(Dijkema, et al., EMBO J. 4:761 [1985]). Other examples ofpromoter/enhancer elements active in a broad range of mammalian celltypes are those from the human elongation factor 1 gene (Uetsuki et al.,J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217 [1990]; andMizushima and Nagata, Nucl. Acids. Res., 18:5322 ([990]) and the longterminal repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl.Acad. Sci. USA 79:6777 [1982]) and the human cytomegalovirus (Boshart etal., Cell 41:521 [1985]).

The terms “promoter element” and “promoter” as used herein refer to aDNA sequence that is located at the 5′ end of (i.e., precedes) a gene ina DNA polymer and provides a site for initiation of the transcription ofthe gene into mRNA.

The terms “gene of interest” and “nucleotide sequence of interest” referto any gene or nucleotide sequence, respectively, the manipulation ofwhich may be deemed desirable for any reason by one of ordinary skill inthe art.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes include a promoter, optionallyan operator sequence, a ribosome binding site and possibly othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

A “modification” as used herein in reference to a nucleic acid sequencerefers to any change in the structure of the nucleic acid sequence.Changes in the structure of a nucleic acid sequence include changes inthe covalent and non-covalent bonds in the nucleic acid sequence.Illustrative of these changes are mutations, mismatches, strand breaks,as well as covalent and non-covalent interactions between a nucleic acidsequence (which contains unmodified and/or modified nucleic acids) andother molecules. Illustrative of a covalent interaction between anucleic acid sequence and another molecule are changes to a nucleotidebase (e.g., formation of thymine glycol) and covalent cross-linksbetween double-stranded DNA sequences which are introduced by, forexample, ultraviolet radiation or by cis-platinum. Yet another exampleof a covalent interaction between a nucleic acid sequence and anothermolecule includes covalent binding of two nucleic acid sequences topsoralen following ultraviolet irradiation. Non-covalent interactionsbetween a nucleic acid sequence and another molecule includenon-covalent interactions of a nucleic acid sequence with a moleculeother than a nucleic acid sequence and other than a polypeptidesequence. Non-covalent interactions between a nucleic acid sequence witha molecule other than a nucleic acid sequence and other than apolypeptide sequence are illustrated by non-covalent intercalation ofethidium bromide or of psoralen between the two strands of adouble-stranded deoxyribonucleic acid sequence. The present inventioncontemplates modifications which cause changes in a fuinctional property(or properties), such changes manifesting themselves at a variety oftime points.

The term “allelic series” when made in reference to a gene refers towild-type sequences of the gene. An “allelic series of modifications” asused herein in reference to a gene refers to two or more nucleic acidsequences of the gene, where each of the two or more nucleic acidsequences of the gene contains at least one modification when comparedto the wild-type sequences of the gene.

As used herein, the term “mutation” refers to a deletion, insertion, orsubstitution. A “deletion” is defined as a change in a nucleic acidsequence in which one or more nucleotides is absent. An “insertion” or“addition” is that change in a nucleic acid sequence which has resultedin the addition of one or more nucleotides. A “substitution” resultsfrom the replacement of one or more nucleotides by a molecule which is adifferent molecule from the replaced one or more nucleotides. Forexample, a nucleic acid may be replaced by a different nucleic acid asexemplified by replacement of a thymine by a cytosine, adenine, guanine,or uridine. Alternatively, a nucleic acid may be replaced by a modifiednucleic acid as exemplified by replacement of a thymine by thymineglycol.

The term “mismatch” refers to a non-covalent interaction between twonucleic acids, each nucleic acid residing on a different polynucleicacid sequence, which does not follow the base-pairing rules. Forexample, for the partially complementary sequences 5′-AGT-3′ and5′-AAT-3′, a G-A mismatch is present.

The terms “nucleic acid” and “unmodified nucleic acid” as used hereinrefer to any one of the known four deoxyribonucleic acid bases (i.e.,guanine, adenine, cytosine, and thymine). The term “modified nucleicacid” refers to a nucleic acid whose structure is altered relative tothe structure of the unmodified nucleic acid. Illustrative of suchmodifications would be replacement covalent modifications of the bases,such as alkylation of amino and ring nitrogens as well as saturation ofdouble bonds.

The term “modified cell” refers to a cell which contains at least onemodification in the cell's genomic sequence.

The term “nucleic acid sequence-modifying agent” refers to an agentwhich is capable of introducing at least one modification into a nucleicacid sequence. Nucleic acid sequence-modifying agents include, but arenot limited to, chemical compounds (e.g., N-ethyl-N-nitrosurea (ENU),methylnitrosourea (MNU), procarbazine hydrochloride (PRC), triethylenemelamine (TEM), acrylamide monomer (AA), chlorambucil (CHL), melphalan(MLP), cyclophosphamide (CPP), diethyl sulfate (DES), ethyl methanesulfonate (EMS), methyl methane sulfonate (MMS), 6-mercaptopurine (6MP),mitomycin-C (MMC), procarbazine (PRC),N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ³H₂O, and urethane (UR)),and electromagnetic radiation (e.g., X-ray radiation, gamma-radiation,ultraviolet light).

The term “wild-type” when made in reference to a gene refers to a genewhich has the characteristics of that gene when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and/or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

A “variant of CNG” is defined as an amino acid sequence which differs byone or more amino acids from the wild-type CNG sequence. The variant mayhave conservative changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have nonconservative changes(e.g., replacement of a glycine with a tryptophan). Similar minorvariations may also include amino acid deletions or insertions (i.e.,additions), or both. A variant CNG can be engineered using any number ofmolecular biology techniques known in the art including but not limitedtosite-directed mutagenesis of a CNG clone. Variant CNG channelssuitable for use with the methods and compositions of the presentinvention include but are not limited to homologues of the rat olfactoryCNG channel (e.g., CNG channles of other species such as mouse and man).

The term “conservative substitution” as used herein refers to a changethat takes place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids can be divided intofour families: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17–21, 2nd ed, WH Freeman and Co. [1981]). Whether achange in the amino acid sequence of a peptide results in a functionalhomolog can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner. In contrast, the term “nonconservative substitution”refers to a change in which an amino acid from one family is replacedwith an amino acid from another family (e.g., replacement of a glycinewith a tryptophan). Guidance in determining which amino acid residuescan be substituted, inserted, or deleted without abolishing biologicalactivity can be found using computer programs (e.g., LASERGENE software,DNASTAR Inc., Madison, Wis.).

The terms “targeting vector” and “targeting construct” are usedinterchangeably to refer to oligonucleotide sequences comprising a geneencoding a cyclic nucleotide gated ion channel. It is preferred that thetargeting vector also comprise a selectable marker gene. The targetingvector contains gene sequences sufficient to permit the homologousrecombination of the targeting vector into at least one allele of thegene resident in the chromosomes of the target or recipient cell (e.g.,ES) cells. Typically, though not necessarily, the targeting vectorcontains 2 kb to 10 kb of DNA homologous to the gene. This 2 kb to 10 kbof DNA maybe located downstream or upstream of the selectable markergene, or may be divided on each side of the selectable marker gene. In apreferred embodiment, the selectable marker gene is located upstream ofthe gene. The targeting vector may contain more than one selectablemaker gene. When more than one selectable marker gene is employed, thetargeting vector preferably contains a positive selectable marker (e.g.,the neo gene) and a negative selectable marker (e.g., the diphtheriatoxin (dt gene) or Herpes simplex virus tk (HSV-tk) gene). The presenceof the positive selectable marker permits the selection of recipientcells containing an integrated copy of the targeting vector whether thisintegration occurred at the target site or at a random site. Thepresence of the negative selectable marker permits the identification ofrecipient cells containing the targeting vector at the targeted site(i.e., which has integrated by virtue of homologous recombination intothe target site); cells which survive when grown in medium which selectsagainst the expression of the negative selectable marker do not containa copy of the negative selectable marker.

In some embodiments, the targeting vectors of the present invention areof the “replacement-type;” integration of a replacement-type vectorresults in the insertion of a selectable marker into the target gene.Replacement-type targeting vectors may be employed to disrupt a generesulting in the generation of a null allele (i.e., an allele incapableof expressing a functional protein; null alleles may be generated bydeleting a portion of the coding region, deleting the entire gene,introducing an insertion and/or a frameshift mutation, etc.) or may beused to introduce a modification (e.g., one or more point mutations)into a gene.

The terms “selectable marker” or “selectable gene product” as usedherein refer to the use of a gene which encodes an enzymatic activitythat confers resistance to an antibiotic or drug upon the cell in whichthe selectable marker is expressed. Selectable markers may be“positive”; positive selectable markers typically are dominantselectable markers (i.e., genes which encode an enzymatic activity whichcan be detected in any mammalian cell or cell line [including EScells]). Examples of dominant selectable markers include, but are notlimited to, (1) the bacterial aminoglycoside 3′ phosphotransferase gene(also referred to as the neo gene) which confers resistance to the drugG418 in mammalian cells, (2) the bacterial hygromycin Gphosphotransferase (hyg) gene which confers resistance to the antibiotichygromycin and (3) the bacterial xanthine-guanine phosphoribosyltransferase gene (also referred to as the gpt gene) which confers theability to grow in the presence of mycophenolic acid. Selectable markersmay be “negative”; negative selectable markers encode an enzymaticactivity whose expression is cytotoxic to the cell when grown in anappropriate selective medium. For example, the HSV-tk gene and the dtgene are commonly used as a negative selectable marker. Expression ofthe HSV-tk gene in cells grown in the presence of gancyclovir oracyclovir is cytotoxic; thus, growth of cells in selective mediumcontaining gancyclovir or acyclovir selects against cells capable ofexpressing a functional HSV TK enzyme. Similarly, the expression of thedt gene selects against cells capable of expressing the diphtheriatoxin;

An animal whose genome “comprises a heterologous selectable marker gene”is an animal whose genome contains a selectable marker gene notnaturally found in the animal's genome which is introduced by means ofmolecular biological methods. A heterologous selectable marker isdistinguished from an endogenous gene naturally found in the animal'sgenome in that expression or activity of the heterologous selectablemarker can be selected for or against.

The terms “in operable combination,” “in operable order,” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a nucleic acid sequence comprising thecoding region of a gene (i.e. the nucleic acid sequence which encodes agene product). The coding region may be present in either a cDNA,genomic DNA or RNA form. When present in a DNA form, the oligonucleotidemay be single-stranded (i.e., the sense strand) or double-stranded.Suitable control elements (e.g., enhancers, promoters, splice junctions,polyadenylation signals, etc.) may be placed in close proximity to thecoding region of the gene, if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers, splicejunctions, intervening sequences, polyadenylation signals, or othersequences, or a combination of both endogenous and exogenous controlelements.

The term “an oligonucleotide sequence comprising at least a portion of acyclic nucleotide gated ion channel gene” refers to a polynucleotidesequence (i.e., a nucleic acid sequence) containing a nucleotidesequence derived from the cyclic nucleotide gated ion channel gene. Thissequence may encode a portion of the cyclic nucleotide gated ion channelgene (i.e., not the entire sequence); alternatively, this sequence mayencode the entire sequence or may simply contain non-coding regionsderived from the gene or a combination of coding and non-coding regions.The oligonucleotide may be RNA or DNA and may be of genomic or syntheticorigin.

As used herein the term “portion” when in reference to a gene refers tofragments of that gene. The fragments may range in size from 10nucleotides to the entire gene sequence minus one nucleotide. Thus, “anoligonucleotide comprising at least a portion of a gene” may comprisesmall fragments of the gene or nearly the entire gene.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”is therefore a substantially purified polynucleotide.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods described in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,965,188, hereby incorporated by reference, which describe methods forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide sequence can be amplified with the appropriate set ofprimer molecules. In particular, the amplified segments created by thePCR process itself are, themselves, efficient templates for subsequentPCR amplifications. Amplified target sequences may be used to obtainsegments of DNA (e.g., genes) for the construction of targeting vectors,transgenes, etc.

As used herein, the terms “PCR product” and “amplification product”refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

The terms “reverse transcription polymerase chain reaction” and “RT-PCR”refer to a method for reverse transcription of an RNA sequence togenerate a mixture of cDNA sequences, followed by increasing theconcentration of a desired segment of the transcribed cDNA sequences inthe mixture without cloning or purification. Typically, RNA is reversetranscribed using a single primer (e.g., an oligo-dT primer) prior toPCR amplification of the desired segment of the transcribed DNA usingtwo primers.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and of an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that it is detectable in any detection system, including,but not limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double- orsingle-stranded nucleic acid at or near a specific nucleotide sequence.

The terms “compound” and “drug candidate” refers to any chemical orbiological entity (e.g., including pharmaceuticals, drugs, and the like)that can be used to treat or prevent a disease, illness, sickness, ordisorder of bodily function. Compounds comprise both known and potentialtherapeutic compounds. A compound can be determined to be therapeutic byscreening, e.g., using the screening methods of the present invention. A“known therapeutic compound” refers to a therapeutic compound that hasbeen shown (e.g., through animal trials or prior experience withadministration to humans) to be effective in such treatment orprevention.

A compound is said to be “in a form suitable for administration” whenthe compound may be administered to an animal by any desired route(e.g., oral, intravenous, subcutaneous, intramuscular, etc.) and thecompound or its active metabolites appear in the desired cells, tissueor organ of the animal in an active form.

As used herein, the term “therapeutic amount” refers to that amount of acompound required to neutralize undesirable pathologic effects in asubject.

As used herein, the term “C460W mutation” refers to the replacement ofcysteine residue with a tryptophan residue at position 460 of the aminoacid sequence of the rat olfactory cyclic nucleotide-gated ion channel.Similar mutations are contemplated to be useful in the context of thepresent invention, including but not limited to C460F and C460Ysubstitutions. In fact, any mutation(s) which also serves to decreasethe NO sensitivity of the channel and increase the cAMP sensitivity ofthe channel find use with the methods and compositions of the presentinvention. Decreased NO sensitivity of a mutant channel can bedetermined by examining channel function in the presence and absence ofa NO donor such as S-nitrosocysteine using published methods (Broillet,J. Biol. Chem., 275:15135–15141 [2000]), and increased cAMP sensitivityof a mutant channel can be determined by generating dose response curvesfor cAMP using the methods disclosed herein and in Rich et al. (J. Gen.Physiol., 118:63–77 [2001]).

As used herein, the term “E583M mutation” refers to the replacement ofthe glutamic acid residue with a methionine residue at position 583 ofthe amino acid sequence of the rat olfactory cyclic nucleotide-gated ionchannel. Similar mutations are contemplated to be useful in the contextof the present invention, including but not limited to E583V, E583L, andE583I substitutions. In fact, any mutation(s), which also serves toincrease cAMP sensitivity and/or decrease cGMP sensitivity finds usewith the methods and compositions of the present invention. IncreasedcAMP sensitivity and/or decreased cGMP sensitivity of a mutant channelcan be determined by generating dose response curves for cAMP and cGMP,respectively using the methods disclosed herein and in Rich et al. (J.Gen. Physiol., 118:63–77 [2001]).

As used herein, the term “Δ61-90 mutation” refers to the deletion ofresidues 61–90 of the amino acid sequence of the rat olfactory cyclicnucleotide-gated ion channel. Other deletion(s), insertion(s) orsubstitution(s) which decrease the sensitivity of the channel toCa2+-CaM regulation find use with the methods and compositions of thepresent invention. Decreased Ca²⁺-CaM sensitivity of a mutant channelcan be determined by examining binding of radiolabeled CaM to mutantchannels and measuring electric currents through the mutant channel inthe presence of Ca²⁺-CaM using published methods (Liu et al., Science,266:1348–1354 [1994]).

Importantly, channels with mutations in regions outside of thosedisclosed herein, are also contemplated to find use with the tools andtechniques of the present invention

The term “stimulus” as used herein refers to any substance or agentwhich excites or produces a temporary increase of vital action in eitherin a whole cell or in any of its parts. Preferred “stimuli” are thosewhich directly or indirectly alter cAMP levels in a cell or in acompartment of a cell.

The term “adenylate cyclase activator” as used herein, refers tocompounds such as forskolin which are capable of activating theadenylate cyclase system and the biosynthesis of cAMP. “Forskolin” isderived from the plant coleus forskohlii.

The term “prostaglandin” as used herein, refers to any of any of a groupof components derived from unsaturated 20 carbon fatty acids, primarilyarachidonic acid, via the cyclooxygenase pathway that are extremelypotent mediators of a diverse group of physiologic processes. Theabbreviation for prostaglandin is PG, specific compounds are designatedby adding one of the letters A through I to indicate the type ofsubstituents found on the hydrocarbon skeleton and a subscript (1, 2 or3) to indicate the number of double bonds in the hydrocarbon skeletonfor example, PGE₂. The predominant naturally occurring prostaglandinsall have two double bonds and are synthesised from arachidonic acid (5,8, 11, 14 eicosatetraenoic acid). The 1 series and 3 series are producedby the same pathway with fatty acids having one fewer double bond (8,11, 14 eicosatrienoic acid) or one more double bond (5, 8, 11, 14, 17eicosapentaenoic acid) than arachidonic acid. All of the prostaglandinsact by binding to specific cell surface receptors causing an increase inthe level of the intracellular second messenger cAMP (and in some casescyclic GMP also).

As used herein, the terms “G-protein” and “GTP protein” refer to any oneof a group of intracellular membrane associated proteins with a highaffinity for guanine nucleotides, and which serve as second messengersor transducers of the receptor-initiated response to intracellularelements such as enzymes to initiate an effect. They are also mediatorsof activated cell-surface receptors and their enzymes, or of ionchannels. G-proteins are responsible for activating a chain of eventsthat alter the concentration of intracellular signaling molecules suchas cAMP and calcium. Thus, the term “G-protein activator” refers tocompounds such as cholera toxin, which are capable of activatingG-proteins.

The term “phosphodiesterase” as used herein refers to an enzyme thatcleaves phosphodiesters to give a phosphomonoester and a free hydroxylgroup. In preferred embodiments, the term is used to refer to enzymessuch as cAMP phosphodiesterase, that convert cyclic nucleotides to themonoester forms. Thus, the term “phosphodiesterase inhibitor” refers tocompounds such as rolipram, which inhibit or antagonize the biosynthesisor actions of a phosphodiesterase.

As used herein, the term “dose response curve” refers to a graphdepicting the relationship between the dose of a drug or other chemical(e.g., cAMP) and the degree of response it produces (e.g., electriccurrent).

The term “electric current” as used herein refers to the rate of chargeflow past a given point in an electric circuit, measured in amperes(coulombs/second).

As used herein, the term “fluorescent Ca2+ indicator” refers tocompounds used to probe Ca2+ concentration via their fluorescentspectral changes upon Ca2+ binding. Fluorescent calcium indicatorsfinding use with the methods and compositions of the present inventioninclude but are not limited to Fura-2, Indo-1, Fluo-3 and Rhod-2.

The term “local intracellular cAMP concentration” refers to the cAMPconcentration detected at a restricted or limited part of the cell(e.g., membrane localized). In contrast, the term “total intracellularcAMP concentration” refers to the cAMP concentration detected throughoutthe cell (e.g., entire accumulated amount divided by the accessiblevolume of the cell).

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: C (degrees Centigrade); rpm (revolutions perminute); BSA (bovine serum albumin); CFA (complete Freund's adjuvant);IFA (incomplete Freund's adjuvant); IgG (immunoglobulin G); IM(intramuscular); IP (intraperitoneal); IV (intravenous orintravascular); SC (subcutaneous); H₂O (water); HCl (hydrochloric acid);aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons);gm (grams); μg (micrograms); mg (milligrams); ng (nanograms); μl(microliters); ml (milliliters); mm (millimeters); nm (nanometers); μm(micrometer); M (molar); mM (millimolar); μM (micromolar); U (units); V(volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes);hr(s) (hour/hours); w/v (weight to volume); v/v (volume to volume);MgCl₂ (magnesium chloride); NaCl (sodium chloride); NO (nitric oxide);OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (optical density at 600 nm); AC(adenylyl cyclase); AKAP (A-kinase anchoring protein); HEK (humanembryonic kidney); NPE-cAMP (1-(2-nitrophenyl)ethyl-cAMP); oCNG channel(olfactory cyclic nucleotide-gated channel); PDE (phosphodiesterase);CaM (calmodulin); IBMX (3-isobutyl-1-methylxanthine); pCPT-cGMP(8-p-chlorophenylthio-cGMP; RO-20-1724(4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone); VIP (vasoactiveintestinal peptide); WT (wild-type); Tris (tris(hydroxymethyl)aminomethane); SDS (sodium dodecyl sulfate); PAGE (polyacrylamide gelelectrophoresis); SDS PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mMsodium phosphate buffer, pH 7.2]); PEG (polyethylene glycol); PMSF(phenylmethylsulfonyl fluoride); PCR (polymerase chain reaction); RT-PCR(reverse transcription PCR); Molecular Probes (Molecular Probes, Eugene,Oreg.); Calbiochem (Calbiochem-Novabiochem Corp., San Diego, Calif.);Amersham (Amersham Pharmacia Biotech, Piscataway, N.J.); ICN (ICNPharmaceuticals, Inc., Costa Mesa, Calif.); ATCC (American Type CultureCollection, Rockville, Md.); BioRad (BioRad, Richmond, Calif.); Clontech(CLONTECH Laboratories, Palo Alto, Calif.); Life Technologies, GIBCOBRL, and Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.);Invitrogen (Invitrogen Corp., San Diego, Calif.); New England Biolabs(New England Biolabs, Inc., Beverly, Mass.); Novagen (Novagen, Inc.,Madison, Wis.); Sigma (Sigma Aldrich, St. Louis, Mo.); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); Gemini (GeminiBioproducts, Calabassas, Calif.); and Hi-Tech (Hi-Tech, Salisbury,United Kingdom). Mathworks (Mathworks, Natick, Mass.); Axon Instruments(Axon Instruments, Foster City, Calif.); Perkin-Elmer (Norwalk, Conn.);and Warner Instruments (Warner Instruments (Warner Instruments, Hamden,Conn.).

Data in the accompanying Figures are representative of at least fourexperiments. Unless otherwise indicated, all experiments were performedat room temperature (19–22° C.). Forskolin and PDE inhibitors were fromCalbiochem. Fura-2/AM and pluronic F-127 were from Molecular Probes.[2-³H]Adenine, [³H]cAMP, and [α-³²P]ATP were from Amersham. All otherchemicals were from Sigma.

Example 1 Cell Culture and Channel Expression

Human embryonic kidney (HEK-293) cells were maintained in culture andinfected with adenovirus as known in the art (See, Rich et al. [2001],supra). As described in greater detail in Example 2, an adenovirusencoding the ax subunit of the rat olfactory CNG channel (CNG2, CNC3)with mutations C460W and E583M was constructed using the Quik ChangeSite-Directed Mutagenesis Kit (Stratagene) and a modification of theAdEasy system (He et al., Proc. Natl. Acad. Sci. USA 95:2509–2514[1998]; and Orlicky and Schaack, J. Lipid Res., 42:460–466 [2001]). Allcells used in cAMP assays were treated with this adenovirus.

Briefly, HEK-293 were maintained in MEM (Life Technologies Inc.)supplemented with 26.2 mM NaHCO₃, 10% (v/v) fetal bovine serum (Gemini),penicillin (50 μg/mL), and streptomycin (50 μg/mL), pH 7.0, at 37° C. ina humidified atmosphere of 95% air and 5% CO₂. Cells were plated at ˜60%confluence in 100 mm culture dishes twenty-four hours prior to infectionwith the CNG-channel-encoding adenovirus constructs (multiplicity ofinfection=10 plaque forming units per cell). Two hours post-infection,hydroxyurea was added to the cell media at 2 mM final concentration topartially inhibit viral replication. Twenty-four hours post-infectioncells were detached with phosphate-buffered saline containing 0.03%EDTA, resuspended in serum-containing medium, and assayed within 12hours.

GH4C1 rat pituitary cells (ATCC) were maintained in 13 mL Ham's F-10medium (Life Technologies) supplemented with 14.3 mM NaHCO₃, 15% donorhorse serum (Gemini), and 2.5% fetal bovine serum, pH 6.8, in 75 cm²flasks at 37° C. in a humidified atmosphere of 95% air and 5% CO₂. Cellswere split weekly (1:4) and washed with fresh medium twice weekly. Cellswere plated at ˜60% confluence in 100 mm culture dishes twenty-fourhours prior to infection with the CNG-channel-encoding adenovirusconstructs (multiplicity of infection=50 plaque forming units per cell).Forty-eight hours post-infection cells were detached, resuspended inserum-containing medium, and assayed within 12 hours.

Example 2 Construction of CNG-Channel-Encoding Adenoviruses

In this Example, the methods used to construct CNG-channel encodingadenoviruses are described. As indicated above in Example 1, pointmutations were introduced into the α subunit of the WT rat olfactory CNGchannel (CNG2, CNC3) using the QuikChange Site-Directed Mutagenesis Kit(Stratagene). Overlap extension PCR was used to delete the Ca²⁺-CaMbinding site (amino acids 61–90). A replication-defective adenovirus, inwhich the channel coding sequence containing the E583M mutation replacedthe E1 region, was constructed as known in the art and describedpreviously (Fagan et al., J. Biol. Chem., 274:12445–12453 [2000]).Briefly, the cDNA encoding the mutant channel was ligated into theplasmid pACCMV (See, Gomez-Foix et al., J. Biol. Chem.,267:25129–25134[1992]) under the control of the cytomegalovirus majorimmediate early (CMV) promoter between the BamHI and SalI sites. Theplasmid was then digested with SalI and ligated with a BstBI adaptor.The resultant plasmid was then digested with BstBI and XmnI and ligatedwith BstBI-digested Ad5dl327_(Bst)-gal-terminal-protein complex, thathad been isolated by banding purified Ad5dl327_(Bst)-gal (Schaack etal., J. Virol., 69:3920–3923 [1995]) virions in 2.8 M CsCl, 4 Mguanidine-HCl. The ligation products were used to transfect HEK-293cells using Ca₃(PO₄)₂ precipitation (Jordan et al., Nucl. Acids Res.,24:596–601 [1996]).

The transfected cells were incubated for seven days. The cells werefrozen and thawed to release virus, and dilutions used to infect HEK-293plates for plaque purification. The infected HEK-293 plates wereoverlaid with medium in Noble agar, fed after 4 days, and stained withX-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) and neutral red.Clear plaques, which were derived from viral chromosomes lacking theLacZ gene of the parental virus, were amplified and analyzed by PCR andrestriction digestion for the presence of the mutated CNG channel cDNA.

Adenovirus transducing vectors encoding C460W/E583M andΔ61-90/C460W/E583M channels were constructed using the AdEasy system (Heet al., Proc. Natl. Acad. Sci. USA 95:2509–2514 [1998]). The cDNAencoding the mutant channel was ligated into pShuttle-CMV between theKpnI and XbaI sites. The resultant plasmid was then linearized bydigestion with PmeI and used to transform E. coli strain BJ5183 that hadbeen transformed with pAdEasy-1. A plasmid containing the adenoviruschromosome encoding the mutated CNG channel was digested with PacI torelease the adenovirus chromosome, and this DNA was used to transfectHEK-293 cells. After incubation for seven days, the virus was releasedby freezing and thawing, and plaque purified. The purified virus wastested for the presence of the CNG channel cDNA by PCR. A viruscontaining the channel cDNA was grown in large scale in HEK-293 cellsand purified by banding using CsCl step and isopycnic gradients.

Example 3 Assessment of cAMP Sensitivity of CNG Channel Constructs

To assess the cyclic nucleotide sensitivity of different CNG channelconstructs, excised, inside-out patch recordings were made at roomtemperature (20–21° C.) using an Axopatch-200A patch clamp amplifier(Axon Instruments Inc.). Pipettes were pulled from borosilicate glassand heat polished. Pipettes were lowered onto the cells and gigaohmseals were formed. Patches were excised by shearing cells from thepipette with a jet of liquid. Ionic currents were elicited by 250 mspulses to membrane potentials of +50 and −50 mV from a holding potentialof 0 mV. Current records were sampled at five times the filter settingand stored on an IBM compatible computer. Records were corrected forerrors due to series resistance (pipette resistance was 4.1±0.1MΩ). Boththe pipette and bath solutions contained (mM): 130 NaCl, 2 HEPES, 0.02EDTA, and 1 EGTA, pH 7.6. Cyclic nucleotide-induced currents wereobtained from the difference between currents in the presence andabsence of cyclic nucleotides. Dose response curves for cAMP and cGMPwere obtained at +50 and −50 mV in the same patch. The effects of themodifications were assessed using the Hill equation,I/I_(max)=[cNMP]^(N)/([cNMP]^(N)+K_(1/2) ^(N)), where I/I_(max) is thefraction of maximal current, cNMP represents cyclic nucleotide, K_(1/2)is the concentration that gives half-maximal current, and N is the Hillcoefficient, an index of cooperativity. K_(1/2) and N for cAMP were 36±5μM, 10±2 μM, 1.0±0.3 μM, and 11±1 μM for WT, E583M, C460/E583M, andΔ61-90/C460W/E583M channels, respectively (See, Rich et al., J. Gen.Physiol. [2001], supra).

Example 4 Detection of Local cAMP in Cell Populations

Increases in local cAMP concentration activate CNG channels and triggerCa²⁺ entry. Ca²⁺ influx was monitored in cell populations using thefluorescent indicator fura-2. Cells were loaded with 4 μM fura-2/AM (themembrane permeant form) and 0.02% pluronic F-127 for 30–40 min, in abuffer containing Ham's F-10 medium supplemented with 1 mg/mL BSA and 20mM HEPES, pH 7.4. In some experiments, HEK-293 cells were loaded with ahigher concentration of fura-2/AM (16 μM) for comparison.

Cells were washed twice, resuspended in the buffer described above(3–4×10⁶ cells/3 mL buffer solution), resuspended in a solutioncontaining (mM): 145 NaCl, 11 D-Glucose, 10 HEPES, 4 KCl, 1 CaCl₂, 1MgCl₂, and 1 mg/mL BSA, pH 7.4 (3–4×10⁶ cells/3 mL solution), andassayed using an LS-50B spectrofluorimeter (Perkin Elmer). Additionswere made by pipetting stock solutions into a stirred cuvette. Thesolutions were then assayed using an LS-50B spectrofluorimeter (PerkinElmer). Additions were made by pipetting stock solutions into a stirredcuvette (mixing time˜5 s).

Fluorescence was measured at an excitation wavelength of 380 nm and anemission wavelength of 510 nm. Under these conditions Ca²⁺ influx wasfound to cause a decrease in fluorescence (ΔF), which was expressedrelative to the pre-stimulus fluorescence (F₀) to correct for variationsin dye concentration, and to allow for comparison of results ondifferent batches of cells. ΔF/F₀ was plotted with inverted polarity, sothat increases in Ca²⁺ influx were represented as positive deflections.Linear fits to the steady-state Ca²⁺ influx rates were used to quantifythe results (Rich et al. [2001], supra). The MATLAB software package(MathWorks) was used for curve fitting and simulations.

Data were sampled at 0.5 Hz and filtered at 0.1 Hz (See FIG. 7, Panels Aand B) or sampled at 6 Hz and filtered at 1.2 Hz (See, FIG. 7, Panels Cand D).

The measurement of absolute Ca²⁺ influx rates using fluorescencerequires that fura-2 overwhelm endogenous Ca²⁺ buffers, and that therelation between fluorescence changes and Ca²⁺ entry be known(Schneggenburger et al., [1993]; Frings et al., [1995]). However, inthese experiments, the concern was the relative Ca²⁺ influx rates, whichreport changes in cAMP levels. This requires only that fura-2 detect afixed proportion of the entering Ca²⁺ in a given experiment. For this tobe true, the concentration of unbound fura-2 should not changeappreciably upon Ca²⁺ binding, and cellular Ca²⁺ buffers that are notoverwhelmed by fura-2 should also not be significantly depleted by Ca²⁺binding. Under these conditions, the equilibrium concentration ofCa²⁺-bound fura-2 (CaF), monitored by fluorescence at 380 nm, is givenby:

$\begin{matrix}{\lbrack{CaF}\rbrack = \frac{\left\lbrack {Ca}_{T} \right\rbrack}{1 + \frac{K_{F}}{\lbrack F\rbrack} + {\frac{K_{F}}{\lbrack F\rbrack} \cdot \frac{\lbrack B\rbrack}{K_{B}}}}} & \left( {{Eq}.\; 6} \right)\end{matrix}$where F represents fura-2, K_(F) the dissociation constant for Ca²⁺binding to fura-2, B the endogenous buffer, K_(B) the dissociationconstant for Ca²⁺ binding to the endogenous buffer, and Ca_(T) the totalCa²⁺ concentration that freely exchanges between fura-2 and theendogenous buffer. The equilibrium assumption is reasonable given thetime scale of the experiments presented here (tens to hundreds ofseconds) and the time scale of binding and unbinding of Ca²⁺ from fura-2and endogenous buffers (milliseconds). Eq. 6 indicates that with [F] and[B] unchanged by the binding of Ca²⁺, [CaF] will be a constant fractionof [Ca_(T)], and therefore directly proportional to the amount ofentering Ca²⁺. In experiments in which quantitative information wasextracted (e.g., the in vivo estimate of K_(I) for PDE inhibitors), lowlevels of Ca²⁺ influx were purposely utilized.

Results from a typical experiment are shown in FIG. 12, in whichaddition of 0.5 μM forskolin (an adenylyl cyclase activator) and 50 nMrolipram (a PDE inhibitor) caused a rise in cAMP and an increase in Ca²⁺influx. Additional experiments are described in the following Examples.

There are several lines of evidence that free fura-2 levels did notchange significantly at these low influx rates, and that depletion ofendogenous buffers did not distort the measurements. First, after abrief delay (during which cAMP rose to steady level), the Ca²⁺ influxrate was constant for a substantial period (linear fit in FIG. 12).Second, the changes in fluorescence over which Ca²⁺ influx rates weremeasured were always a very small fraction of the total change influorescence measured when saturating Ca²⁺ was admitted into the cells(by adding 30 μL of 10% Triton to the cuvette). The fluorescence changesused in the linear fits were generally <10% of the saturated fura-2response, indicating that unbound fura-2 was predominant. At high levelsof Ca²⁺ influx, non-linearities in the traces are likely due todepletion of fura-2 and Ca²⁺ pumping mechanisms. Third, increasing theexternal fura-2/AM concentration from 4 to 16 μM significantly alteredthe intracellular fura-2 concentration but had no effect on themeasurement of relative Ca²⁺ influx rates or estimates of PDE inhibitorK_(I). The intracellular concentrations of fura-2 were not determined,but a previous study of neuroblastoma cells and isolated pulmonaryartery endothelial cells found that 60 min loading with 10 μM fura-2/AMyielded intracellular fura-2 concentrations of about 130 μM (Oakes etal., [1988]). If HEK-293 cells behave similarly, they would be expectedto display intracellular concentrations of 35 and 140 μM under the twoloading conditions.

Neher and Augustine (Nether and Augustine [1992]) have shown in adrenalchromaffin cells that 98–99% of entering Ca²⁺ binds to endogenousbuffers that are present at high concentration (375–750 μM) but have alow affinity for Ca²⁺ (K_(B)˜5–10 μM). Thus, the ratio [B]/K_(B) inEquation 6 was estimated to be 75. K_(F) for fura-2 was estimated to be150 nM. Thus, the [F]/K_(F) values under the two loading conditions wereexpected to be 233 and 933. At the higher loading condition, fura-2 maybe overwhelming the low-affinity cellular buffers. However, as pointedout above, this is not necessary as long as the buffers were not beingdepleted. It is very likely that low affinity buffers are present at ahigh enough concentration that they were not depleted by the low amountsof entering Ca²⁺. Two of the lines of evidence cited above indicate thatbuffers (including any high-affinity buffers that may be present) didnot affect the proportion of Ca²⁺ detected by fura-2: the linearity ofthe influx traces (FIG. 12 and the observation that increasing fura-2 bya factor of four does not alter the measurement of relative Ca²⁺ influxrates. These results strongly suggest that any high-affinity bufferswere overwhelmed by the fura-2 concentrations used.

Example 5 In Vivo Identification of PDE Types in HEK-293 Cells Using CNGChannel Constructs

There are over 30 known forms of PDE that have been grouped into 11families. Of these families, PDE types V, VI, and IX are cGMP-specific.Recently, an isoform of PDE type IV, PDE4A, purified from U937 monocyticcells was shown to be inhibited by rolipram with an IC₅₀ of 3 nM(MacKenzie and Houslay Biochem. J., 347:571–578 [2000]). It should benoted that it is contemplated that these efficacies may be modulated invivo. For example, it has been shown that forms of PDE type IVA areapproximately 10-fold more sensitive to rolipram when they bind to theSRC family tyrosyl kinase LYN (McPhee et al., [1999]). Data in Table 3were obtained using heterologously expressed mouse (types VII, VIII, andX) and human (types I, II, IV, V, VII, IX, and XI) PDEs, as well asendogenous PDEs from dog kidney (type IV), rat brain (type IV), bovineheart (types III and IV), human heart (type II), bovine aorta (types I,III, and V), rabbit aorta (types I, IV, and V), and bovine photoreceptor(type VI). Data in Table 3 were compiled from: (1) Ahluawalia and RhoadsBiochem. Pharmacol., 31:665–669 (1982); (2) Ahn et al., Biochem.Pharmacol., 38:3331–3339 (1989); (3) Beavo, Adv. Second MessengerPhosphoprot. Res., 22:1–38 (1988); (4) Bolger et al., Mol. Cell. Biol.,13:6558–6571 (1993); (5) Bolger et al. Biochem. J., 328:539–548 (1997);(6) Coste and Grondin, Biochem. Pharmacol., 50:1577–1585 (1995); (7)Epstein et al., Arch. Biochem. Biophys., 218:119–133 (1982); (8) Fawcettet al., Proc. Natl. Acad. Sci. USA 97:3702–3707 (2000); (9) Fisher etal., J. Biol. Chem., 273:15559–15564 (1998); (10) Gardner et al.,Biochem. Biophys. Res. Commun., 272:186–192 (2000); (11) Harrison etal., Meth. Enzymol., 15:685–702 (1988); (12) Hetman et al., Proc. Natl.Acad. Sci. USA 97:472–476 (2000); (13) Holck et al., J. Cardiovasc.Pharm., 6:520–530 (1984); (14) Lorenz and Wells, Mol. Pharmacol.,23:424–430 (1983); (15) Loughney et al., J. Biol. Chem., 271:796–806(1996); (16) Loughney et aL,Gene 216:139–147 (1998); (17) Nemoz et al.,Biochem. Pharmacol., 15:2997–3000 (1985); (18) Podzuweit et al., CellSignal 7:733–738 (1995); (19) Rosman et al., Gene 191:89–95 (1997); (20)Soderling et al., Proc. Natl. Acad. Sci. USA 95:8991–8996 (1998a); (21)Soderling et aL, J. Biol. Chem., 273:15553–15558 (1998b); (22) Soderlinget al., Proc. Natl. Acad. Sci., 96:7071–7076 (1999); and (23) Whalin etaL, Mol. Pharmacol., 39:711–717 (1991). References cited for variousinhibitors include: IBMX^(3,6,8,10-12,15,16,20-22),8-M-IBBX^(2,4,14,15), EHNA^(12,16,18,22), Trequinsin²³, Quazinone¹³,Etazolate¹, RO-20-1724^(7,9,12,20,21,23), Rolipram^(5,8,9,15,17,20-23),and Zaprinast^(6,8,9,15,16,20-22). Blank spaces in Table 3 indicate thatthese data are not available.

The PDE type(s) that regulates cAMP levels near CNG channels in HEK-293were identified cells using the Ca²⁺ influx assay in cell populations asdescribed in EXAMPLE 3. The C460W/E583M channels were used to monitorforskolin-stimulated cAMP accumulation in the presence and absence of aseries of PDE inhibitors. The reported IC₅₀ values of inhibitors used inthis experiment for each PDE type are given in Table 3. The PDEinhibitor concentrations used were typically at least 5-fold higher thanthe most potent IC₅₀. Either vehicle or PDE inhibitors were added at 0 sand 1 μM forskolin was added at 180 s (See, FIG. 10). In the absence ofPDE inhibitors, little or no forskolin-induced Ca²⁺ influx was observed.However, in the presence of IBMX (10 or 100 μM), or the PDE-type-IVspecific inhibitor RO-20-1724 (10 μM), significant forskolin-inducedCa²⁺ influx was observed (See, FIG. 10, Panels A,B, and G). Two otherPDE type IV inhibitors significantly increased the forskolin-inducedCa²⁺ influx, rolipram (See, FIG. 11, Panel C) and etazolate (data notshown). Inhibitors specific to other PDE families did not affectforskolin-induced Ca²⁺ influx. Similar results were obtained in HEK-293cells expressing Δ61-90/C460W/E583M channels by stimulating cAMPproduction with higher forskolin concentrations (10 μM, data not shown),or when PGE₁ (1 μM) was used to stimulate AC activity (data not shown).In these experiments, inhibitor concentrations were used at whichspecific PDE types should be inhibited. However, many PDE inhibitors arenot completely specific (See, Table 3).

TABLE 3 Specificity of PDE Inhibitors for Different PDE Families RSInhibitor (PDE) I II III IV V VI VII VIII IX X XI IBMX NS 2–3 20–50 2 5–15 4–7 10 2–8 >200 >200 2.6 49.8 8-M-IBBX I 2–8 69 >100 EHNA II0.5–1   >500 >100 >100 >100 69 Trequinsin III 0.5–2   0.0003 QuazinoneIII 0.6 Etazolate IV RO-20-1724 IV   200 >100 52   250 >200 >200Rolipram IV >150 >100 >100 >100 >200 >200 47 >100 Zaprinast V-VI  3-10  70  10 0.2–0.5 0.15 >50  >100 29–35 11 12 NS = Non-selective

Example 6 In Vivo Assessment of PDE Inhibitor K_(I) Using Modified CNGChannels

To further establish that PDE type IV is responsible for the observedIBMX-sensitive PDE activity, the K_(I)'s (inhibition constants) of IBMXand two PDE type IV inhibitors, RO-20-1724 and rolipram were estimatedin vivo. This required developing a quantitative framework to assess therelationship between cAMP synthesis, hydrolysis, and redistributionthroughout the cell. Thus, the following formalism was adopted:

$\begin{matrix}{\frac{\mathbb{d}\lbrack{cAMP}\rbrack}{\mathbb{d}t} = {C - \frac{V_{\max} \cdot \lbrack{cAMP}\rbrack}{{K_{m} \cdot \left( {1 + \frac{\lbrack I\rbrack}{K_{I}}} \right)} + \lbrack{cAMP}\rbrack} - {k_{f} \cdot \lbrack{cAMP}\rbrack}}} & \left( {{Eq}.\mspace{14mu} 4} \right)\end{matrix}$where C is the steady-state rate of cAMP synthesis by AC, V_(max) is themaximal rate of cAMP hydrolysis, K_(m) is the Michaelis constant forPDE, and k_(f) is the rate constant of cAMP flux out of the microdomain.In order to estimate K_(I) for the PDE inhibitors, two assumptions weremade. First, it was assumed that at low levels of AC stimulation and PDEinhibition, the concentration of local cAMP is low and diffusion of cAMPout of the microdomain is negligible. Second, it was assumed that cAMPlevels reach steady-state shortly after AC stimulation (i.e., equalrates of synthesis and hydrolysis). With these assumptions, Equation 4can be simplified to:

$\begin{matrix}{\lbrack{cAMP}\rbrack = {\frac{C \cdot K_{m}}{V_{\max} - C} \cdot \left( {1 + \frac{\lbrack I\rbrack}{K_{I}}} \right)}} & \left( {{Eq}.\mspace{11mu} 5} \right)\end{matrix}$

Interestingly, this equation reveals that when the inhibitorconcentration is equal to K_(I) the cAMP concentration is twice that inthe absence of inhibitor. At low cAMP concentrations (<K_(1/2) for thechannel), the cAMP concentration is proportional to the square root ofthe Ca²⁺ influx rate (the Hill coefficient for channel activation isapproximately 2). Thus, K_(I) can be estimated using a linear fit to thesquare root of the slopes of the fluorescence traces as a function ofinhibitor concentration (See, FIG. 11).

The high cAMP affinity C460W/E583M channels were used to detect changesin cAMP concentration following pretreatment with PDE inhibitors (addedat 0 s) and modest forskolin stimulation (0.5 μM added at 180 s). IBMX,RO-20-1724, and rolipram were completely equilibrated across the plasmamembrane of HEK-293 cells in <180 s (data not shown). The assumptionthat cAMP levels reach steady-state is supported by the long-lastinglinear rise in Ca²⁺ concentration following forskolin stimulation (asteady Ca²⁺ influx rate reflects a constant cAMP level; See, FIG. 12).Dose response relations for the three PDE inhibitors are shown in FIG.11. Based upon fits to this data with Equation 8 (see insets in FIG. 11,Panels A–C), the K_(I) values were estimated to be 11±2 μM (IBMX),0.13±0.02 μM (RO-20-1724), and 0.07±0.02 μM (rolipram), n=4, which areconsistent with published IC₅₀ values from in vitro experiments (See,Table 3). To ensure that the results were independent of channelconstruct the K_(I) for RO-20-1724 was also estimated using the lowercAMP affinity Δ61-90/C460W/E583M channels. The K_(I) (0.15±0.02 μM, n=3)was indistinguishable from that estimated using C460W/E583M channels.These data strongly suggest that PDE type IV is primarily responsiblefor the IBMX-sensitive component of PDE activity monitored using CNGchannels and, thus, for regulating localized cAMP concentration inHEK-293 cells.

Example 7 Measurement of PGE₁-Induced cAMP Responses in HEK-293 CellPopulations

In this Example, experiments conducted to detect cAMP levels in cellsare described. Changes in cyclic AMP concentrations in response to PGE₁stimulation were monitored in HEK-293 cells by measuring Ca²⁺ influxthrough C460W/E583M CNG channels (See, Rich et al. [2001], supra).HEK-293 cells express a variety of extracellular receptors includingprostanoid receptors (Thomas and Hoffman, Mol. Pharmacol., 49:907–914[1996]), and the cAMP-specific PDE type IV (Rich et al [2001], supra;and Hoffman et al., EMBO J., 18:893–903 [1999]). Based on PCR analysis,these cells also appear to express AC types II, III, VI, and VII,(Hellevuo et al., Biochem. Biophys. Res. Commun., 192:311–318 [1993]).C460W/E583M channels were expressed heterologously using an adenovirusconstruct (See, Rich et al. [2001], supra). The Ca²⁺ permeability of thechannel was used to detect changes in local cAMP concentration; thefluorescent indicator fura-2 was used to monitor Ca²⁺ entry. With thisapproach, incremental changes in cAMP concentration are readily detectedas changes in relative Ca²⁺ influx rates through C460W/E583M channels(See, Rich et al., [2001], supra).

FIG. 16 provides a comparison of membrane-localized (A) and total cAMP(B) levels in cell populations. FIG. 16, Panel A, shows that sustainedapplication of 10 μM PGE₁ in the absence of PDE inhibitor caused anincrease in Ca²⁺ influx, followed by a decline in Ca²⁺. Little or noincrease in Ca²⁺ was observed in cells not expressing the channel. Theinitial interpretation was that PGE₁ triggered a rise and fall in localcAMP concentration: the rise caused an increase in Ca²⁺ influx throughCNG channels; the subsequent fall in cAMP led to reduced Ca²⁺ influx;and, Ca²⁺ pumping mechanisms caused the decline in Ca²⁺ levels. Insupport of this interpretation, PGE₁ in the presence of the PDEinhibitor IBMX caused Ca²⁺ to rise along a similar time-course, but thedecay phase was abolished. These results indicate that the underlyingcause of the decay phase was hydrolysis of cAMP. In populations of cellsexpressing C460W/E583M channels, PGE₁ caused total cellular cAMPaccumulation, assessed as the conversion of [³H]ATP to [³H]cAMP, to riseto a plateau in the absence of IBMX (See, FIG. 16, Panel B). This is inmarked contrast to the transient increase in cAMP inferred from FIG. 16,Panel A.

The basis for the transient response was investigated further, as shownin FIG. 17. Transient responses were observed over a large range of PGE₁concentrations, from 0.01 to 10 μM (FIG. 17, Panel A). Forskolin, anactivator of AC, also triggered dose-dependent rises in Ca²⁺ influx, asshown in FIG. 17, Panel B. However, these responses, unlike thePGE₁-induced responses, did not decline. Similarly, using the sameassay, the responses of rat glioma C6-2B cells to isoproterenol and ratpituitary-derived GH4C1 cells to vasoactive intestinal peptide did notdecline on a similar time-scale (See, Rich et al., J. Gen. Physiol.,118:63–77 [2001]; and Fagan et al., FEBS Lett., 500:85–90 [2001]). Thus,the activation of AC alone does not necessarily cause transient Ca²⁺responses. Taken together, these results indicate that the decline wasdue to a reduction in AC activity and/or an increase in PDE activity.The decline was unlikely to result primarily from a reduction in ACactivity (e.g., receptor desensitization), because of the sustainedaccumulation of total cellular cAMP in the presence of IBMX (See, FIG.16, Panel B). To verify this, 100 μM IBMX was added at various timesafter PGE₁ application. Even 50 min after the addition of PGE₁, IBMXtriggered a rapid Ca²⁺ influx, as shown in FIG. 17, Panel C, indicativeof a sharp rise in cAMP level. The type-IV-specific PDE inhibitorRO-20-1724 (10 μM) also caused sharp increases in cAMP (the maximalslopes were indistinguishable from those induced by IBMX).

In the absence of PGE₁, PDE inhibitors caused no measurable Ca²⁺ influx.These experiments suggest that AC activity did not decrease appreciably,but instead that PDE activity was up-regulated. To test whether PGE₁ didindeed increase the total PDE activity, 100 nM PGE₁ were added atvarious times after the initial application of 100 nM PGE₁ (asubsaturating concentration). There was a negligible response to thesubsequent addition of PGE₁, even after 50 min (FIG. 17, Panel D). Takentogether, the results of the experiments shown in FIG. 17, Panels C andD argue that the cAMP transient is due to an initial increase in ACactivity, followed by a more profound increase in PDE activity. Theseresults are consistent with previous studies showing thatneurotransmitters and hormones, including PGE₁, regulate PDE activity aswell as cyclase activity (Trivedi and Kramer, Neuron 21:895–906 [1998];Alvarez et al., Mol. Phannacol., 20:302–309; Macphee et al., J. Biol.Chem., 263:10353–10358 [1988]; Conti et al., Endocrine Rev., 16:370–389[1995]; and Houslay et al., Adv. Pharmacol., 44:225–342 [1998]). Asindicated herein, these processes are crucial for the generation ofspatially and temporally distinct cAMP signals.

Example 8 Measurement of Forskolin- and VIP-Induced Responses in GH4C1Cells

In this Example, experiments conducted to examine the response ofexcitable GH4C1 pituitary cells to stimulation by either forskolin orthe hormone vasoactive intestinal peptide (VIP), in the presence ofabsence of PDE inhibitors are described. In the experiments described, 1μM nimodipine was added at time zero to block Ca²⁺ influx thoughvoltage-gated Ca²⁺ channels (triggered by membrane depolarization due toCa²⁺ and Na⁺ influx through CNG channels). This concentration ofnimodipine was sufficient to block Ca²⁺ influx through voltage-gatedCa²⁺ channels activated by membrane depolarization in 24 mM externalKCl, and did not alter forskolin or pCPT-cGMP induced Ca²⁺ influxthrough CNG channels expressed in HEK-293 cells (data not shown).

FIG. 13, Panel A, shows the forskolin-induced responses of cellsexpressing WT channels. Either vehicle or 100 μM IBMX was added at 0 sand 50 μM forskolin was added at 180 s. After the addition of forskolin,there was a short delay followed by an increased Ca²⁺ influx. The slopeof the Ca²⁺ influx was greater in the presence of IBMX, indicatinghigher cAMP levels. In control cells (i.e., cells not expressing CNGchannel constructs), no forskolin-induced Ca²⁺ influx was observed ineither the presence or absence of IBMX (See, FIG. 13, Panel B). This wastrue of controls done for all experimental protocols.

Next, forskolin-induced responses and the effects of IBMX in cellsexpressing the high cAMP affinity construct, C460W/E583M were examined.In these experiments either vehicle or 100 μM IBMX was added at 60 s(See, FIG. 13, Panels C and D). Interestingly, the addition of IBMXtriggered Ca²⁺ influx that was not observed in either control cells orcells expressing the WT channel. Subsequent addition of 10 μM forskolin(See, FIG. 13, Panel C) or 100 nM VIP (See, FIG. 13, Panel D) causedadditional cAMP accumulation and Ca²⁺ influx. A comparison of responsesmeasured with the WT and C460W/E583M channels indicated that theIBMX-induced response was not due to an increase in local cGMPconcentration. Moreover, 100 μM IBMX did not alter CNG channel activitymonitored in excised patches (data not shown). Thus, these data indicatethat the IBMX-induced Ca²⁺ influx was due primarily to an increase incAMP arising from basal AC activity.

Example 9 Measurement of Local cAMP in Single Cells

Single-cell cAMP measurements were made using either the perforatedpatch or whole cell patch clamp technique (Rich et al. [2000], supra).In the perforated patch configuration, the pore forming antibioticnystatin was added to the pipette solution to gain electrical access tothe cell's interior while retaining divalent cations and largermolecules like cAMP in the cell. Recordings were made using anAxopatch-200A patch clamp amplifier (Axon Instruments). Pipetteresistance was limited to 5 ΩM and averaged 3.4±0.5 ΩM (measurementsprovided herein are expressed as mean±standard deviation). In theperforated patch configuration, a steady access resistance was obtained5–15 minutes following seal formation. Capacitive transients wereelicited by applying −30 mV steps from the holding potential of −20 mVfor calculation of access resistance (100±40 MΩ). These quantities weremonitored throughout the experiments to ensure stable electrical accesswas maintained. Current records were typically sampled at five times thefilter setting. Records were digitally filtered at 12 Hz, resampled at60 Hz, and corrected for errors due to series resistance. The controlbath solution contained (mM): 140 NaCl, 4 KCl, 11 glucose, 10 HEPES, andeither 0.1 or 10 MgCl₂, pH 7.4. Solutions were applied using the SF-77Bfast-step solution switcher (Warner Instruments). The mechanical switchtime was 1 ms. The time to exchange the extracellular solution wasmeasured by applying a 140 mM KCl solution to a depolarized cell (+50mV), and monitoring changes in current through endogenous voltage-gatedK⁺ channels; for each experiment, it was less than 60 ms. The pipettesolution in perforated patch experiments contained 70 mM KCl, 70 mMpotassium gluconate, 4 mM NaCl, 0.5 mM MgCl₂, 10 mM HEPES, pH 7.4, and50–200 μg/ml nystatin. In most of these experiments, the pipettesolution also contained 1 mM cAMP; at the end of the experiment themaximal cAMP-induced current could be measured by rupturing the cellmembrane at the tip of the pipette with suction, allowing saturatingcAMP to diffuse to the channels (See, Rich et al. [2000], supra). Inwhole cell experiments, 5 mM K₂ATP and 0.1 mM Na₂GTP were added to thepipette solution, and nystatin was not included.

The cyclic nucleotide sensitivity of the C460W/E583M CNG channel wasassessed in excised, inside-out patches as known in the art (See, Richet al. [2001], supra). K_(1/2) and N were 1.1±0.3 μM and 2.1±0.4 at +50mV; and 1.0±0.3 μM and 2.0±0.3 at −50 mV (n=11). With these values, thecAMP concentration could be calculated from currents measured inperforated patch experiments (Rich et al. Proc. Natl. Acad. Sci. [2001],supra). For example, if I/I_(max) was found to be 0.6, the estimatedcAMP concentration would be 1.2 μM. It should be noted that the lowconcentration of channels expressed in these cells (˜1 nM) did notsignificantly buffer the measured cAMP signals.

The response to PGE₁ in single cells was examined by directly measuringionic currents through C460W/E583M channels with the perforated patchclamp technique. This approach has higher temporal resolution anddynamic range than the Ca²⁺ influx assay, and the response can becalibrated, allowing for accurate measurement of cAMP concentration.These experiments were done in nominally Ca²⁺-free solutions, whichincreased currents through the channels (Ca²⁺ is a permeant blocker),and removed the possibility that Ca²⁺ entry inhibited the channels (See,Finn et al., Ann. Rev. Physiol., 58:395–426 [1996]). The responses oftwo different cells to rapid application of 1 μM PGE₁ are shown in FIG.15, Panels A and B. Inward currents were measured at a holding potentialof −20 mV. The currents were converted to cAMP concentration based onthe channel's dose-response relation determined in excised membranepatches. The cAMP signals were transient, rising more sharply than theydecayed, in general agreement with the cAMP changes inferred from Ca²⁺influx experiments in cell populations. The absence of extracellularCa²⁺ in these experiments, and, as noted earlier, the lack of aPGE₁-stimulated rise in intracellular Ca²⁺ in control cells, indicatethat the transient cAMP responses were not caused by Ca²⁺ feedbackmechanisms. Additional controls are indicated above, in the Descriptionof the Drawings. The shapes of the responses were similar across sixcells, but the kinetics varied (width at half height 87±53 s). Thetime-course in shown in FIG. 15, Panel A was about average for thesingle cell measurements, while the time-course shown in FIG. 15, PanelB (from a different cell) was considerably faster. The average amplitudeof the cAMP signal in five cells was 0.7±0.4 μM. When 100 μM IBMX wasadded after 100 nM PGE₁ (See, FIG. 15, Panel C), the current rose to aplateau (n=4). This demonstrates the persistent activation of AC byPGE₁, as observed in cell populations (See, FIG. 17, Panel C).

All publications and patents mentioned in the above Specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, genetics, and/or related fields are intended to bewithin the scope of the present invention.

1. An isolated nucleic acid encoding a modified mammalian olfactorycyclic nucleotide-gated ion channel alpha subunit, wherein said channelcomprises a mutation, wherein said mutation comprises a substitution ata residue corresponding to position 583 of SEQ ID NO:8, and wherein saidchannel has increased cAMP sensitivity, and decreased cGMP sensitivityas compared to a wild type channel.
 2. The nucleic acid of claim 1,wherein said substitution at a residue corresponding to position 583 ofSEQ ID NO:8 is selected from the group consisting of E583M, E583V, E583Land E583I.
 3. The nucleic acid of claim 1, wherein said cGMP-inducedcurrent is 40% or less than said cAMP-induced current.
 4. The nucleicacid of claim 1, wherein said increased cAMP sensitivity comprises aK_(1/2) at least ten-fold lower than that observed for a nucleic acidencoding a wild type channel.
 5. The nucleic acid of claim 1, whereinsaid decreased cGMP sensitivity comprises a K_(1/2) at least ten-foldhigher than that observed for a nucleic acid encoding a wild typechannel.
 6. The nucleic acid of claim 1, wherein said mutation furthercomprises a substitution at a residue corresponding to position 460 ofSEQ ID NO:8.
 7. The nucleic acid of claim 6, wherein said substitutionat a residue corresponding to position 460 of SEQ ID NO:8 is selectedfrom the group consisting of C460W, C460F and C460Y.
 8. The nucleic acidof claim 6, wherein said mutation further comprises a 61–90 deletion atresidues corresponding to positions 61–90 of SEQ ID NO:8.
 9. An isolatednucleic acid encoding a modified rat olfactory cyclic nucleotide-gatedion channel alpha subunit, wherein said channel alpha subunit comprisesa glutamic acid (E) to methionine (M) substitution at a residuecorresponding to position 583 of SEQ ID NO:8.
 10. The isolated nucleicacid of claim 9, wherein said channel alpha subunit further comprises acysteine (C) to tryptophan (W) substitution at a residue correspondingto position 460 of SEQ ID NO:8.
 11. The isolated nucleic acid of claim10, wherein said channel alpha subunit further comprises a 61–90deletion at residues corresponding to positions 61–90 of SEQ ID NO:8.12. The isolated nucleic acid of claim 9, wherein said channel comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:7.
 13. An expression vector comprisingthe nucleic acid of claim
 9. 14. The expression vector of claim 13,wherein said vector is a recombinant adenovirus vector.
 15. An isolatedhost cell comprising the expression vector of claim 13, wherein saidhost cell is selected from the group consisting of a prokaryotic celland a eukaryotic cell.
 16. The host cell of claim 15, wherein saideukaryotic cell is selected from the group consisting of a humanembryonic kidney-293 cell and a rat GH4C1 pituitary cell.
 17. Anisolated polypeptide of a modified mammalian olfactory cyclicnucleotide-gated ion channel alpha subunit, wherein said channelcomprises a mutation, wherein said mutation comprises a substitution ata residue corresponding to position 583 of SEQ ID NO:8, and wherein saidchannel has increased cAMP sensitivity, and decreased cGMP sensitivityas compared to a wild type channel.